Structural Variabilities in β-Lactamase (blaA) of Different Biovars of Yersinia enterocolitica: Implications for β-Lactam Antibiotic and β-Lactamase Inhibitor Susceptibilities

Yersiniosis caused by Yersinia enterocolitica has been reported from all continents. The bacterial species is divided into more than fifty serovars and six biovars viz. 1A, 1B, 2, 3, 4 and 5 which differ in geographical distribution, ecological niches and pathogenicity. Most Y.enterocolitica strains harbor chromosomal genes for two β-lactamases, blaA an Ambler class A penicillinase and blaB an Ambler class C inducible cephalosporinase. In the present study, susceptibility to b-lactam antibiotics and β-lactamase inhibitor was studied for Y. enterocolitica strains of biovars 1A, 1B, 2 and 4. We observed that β-lactamases were expressed differentially among strains of different biovars. To understand the molecular mechanisms underlying such differential expression, the sequences of genes and promoters of blaA were compared. Also, the variants of blaA present in different biovars were modeled and docked with amoxicillin and clavulanic acid. The mRNA secondary structures of blaA variants were also predicted in-silico. Our findings indicated that neither variations in the promoter regions, nor the secondary structures of mRNA contributed to higher/lower expression of blaA in different biovars. Analysis of H-bonding residues of blaA variants with amoxicillin and clavulanic acid revealed that if amino acid residues of a β-lactamase interacting with amoxicillin and the clavulanic acid were similar, clavulanic acid was effective in engaging the enzyme, accounting for a significant reduction in MIC of amoxicillin-clavulanate. This finding might aid in designing better β-lactamase inhibitors with improved efficiencies in future.     

Modification of the Susceptibility of Gram-Negative Rods Producing ESβLS to β-Lactams by the Efflux Phenomenon

The production of β-lactamases is the most important mechanism of Gram-negative rod resistance to β-lactams. Resistance to ceftazidime and cefepime in clinical isolates of Enterobacteriaceae (especially ESβL-positive E. coli and K. pneumoniae) and P. aeruginosa is life-threatening. However, all strains of the above mentioned species possess chromosomally encoded RND efflux pump systems in addition to β-lactamase production. The main goal of this study was to assess the role of efflux pump systems in cefepime and/or ceftazidime resistant phenotypes of ESβL-positive clinical strains of Enterobacteriaceae and P. aeruginosa. The influence of the efflux pump inhibitor PAβN on the minimum inhibitory concentration (MIC) values of tested cephalosporins was species-dependent. Generally, a significant reduction (at least four-fold) of β-lactam MICs was observed in the presence of PAβN only in the case of P. aeruginosa clinical isolates as well as the ESβL-producing transformant PAO1161 ΔampC. The usage of this agent resulted in the restoration of susceptibility to cefepime and/or ceftazidime in the majority of the P. aeruginosa ESβL-positive strains with low and moderate resistance to the above cephalosporins. Moreover, an outer membrane permeabilizing effect in the presence of PAβN was identified. Strain-dependent β-lactamase leakage upon PAβN or β-lactam treatment was demonstrated. The most important observation was the restoration of susceptibility of P. aeruginosa WUM226 to cefepime (MIC decrease from 32 to 4 mg/L) and ceftazidime (MIC decrease from 128 to 4 mg/L) in the presence of PAβN, which occurred despite an almost complete lack of β-lactamase leakage from bacterial cells. In conclusion, these data indicate that RND efflux pumps can modify the susceptibility to β-lactams in Gram-negative rods producing ESβLs. However, this phenomenon occurs only in P. aeruginosa strains and was not observed among E. coli and K. pneumoniae strains, representing the Enterobacteriaceae family.     

Characterization of Piperacillin/Tazobactam-Resistant Klebsiella oxytoca Recovered from a Nosocomial Outbreak

We characterized 12 clinical isolates of Klebsiella oxytoca with the extended-spectrum β-lactamase (ESBL) phenotype (high minimum inhibitory concentration [MIC] values of ceftriaxone) recovered over 9 months at a university hospital in Japan. To determine the clonality of the isolates, we used pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and PCR analyses to detect bla _RBI, which encodes the β-lactamase RbiA, OXY-2-4 with overproduce-type promoter. Moreover, we performed the isoelectric focusing (IEF) of β-lactamases, and the determination of the MICs of β-lactams including piperacillin/tazobactam for 12 clinical isolates and E. coli HB101 with pKOB23, which contains bla _RBI, by the agar dilution method. Finally, we performed the initial screening and phenotypic confirmatory tests for ESBLs. Each of the 12 clinical isolates had an identical PFGE pulsotype and MLST sequence type (ST9). All 12 clinical isolates harbored identical bla _RBI. The IEF revealed that the clinical isolate produced only one β-lactamase. E. coli HB101 (pKOB23) and all 12 isolates demonstrated equally resistance to piperacillin/tazobactam (MICs, >128 μg/ml). The phenotypic confirmatory test after the initial screening test for ESBLs can discriminate β-lactamase RbiA-producing K. oxytoca from β-lactamase CTX-M-producing K. oxytoca. Twelve clinical isolates of K. oxytoca, which were recovered from an outbreak at one university hospital, had identical genotypes and produced β-lactamase RbiA that conferred resistance to piperacillin/tazobactam. In order to detect K. oxytoca isolates that produce RbiA to promote research concerning β-lactamase RbiA-producing K. oxytoca, the phenotypic confirmatory test after the initial screening test for ESBLs would be useful.     

A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape

Listeria monocytogenes is a Gram-positive bacterium and a facultative intracellular pathogen that invades mammalian cells, disrupts its internalization vacuole, and proliferates in the host cell cytoplasm. Here, we describe a novel image-based microscopy assay that allows discrimination between cellular entry and vacuolar escape, enabling high-content screening to identify factors specifically involved in these two steps. We first generated L. monocytogenes and Listeria innocua strains expressing a β-lactamase covalently attached to the bacterial cell wall. These strains were then incubated with HeLa cells containing the Förster resonance energy transfer (FRET) probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase only in cells inoculated with L. monocytogenes but not those inoculated with L. innocua, thereby demonstrating bacterial access to the host cytoplasm. Subsequently, we performed differential immunofluorescence staining to distinguish extracellular versus total bacterial populations in samples that were also analyzed by the FRET-based assay. With this two-step analysis, bacterial entry can be distinguished from vacuolar rupture in a single experiment. Our novel approach represents a powerful tool for identifying factors that determine the intracellular niche of L. monocytogenes.     

Analysis of diverse β-lactamases presenting high-level resistance in association with OmpK35 and OmpK36 porins in ESBL-producing Klebsiella pneumoniae

Emerging extensively drug-resistant (XDR) Klebsiella pneumoniae due to the production of β-lactamases and porin loss is a substantial worldwide concern. This study aimed to elucidate the role of outer membrane porin (OMP) loss, AmpC, and carbapenemases among extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae strains with XDR phenotype. This study analyzed 79 K. pneumoniae from several clinical sources and detected ESBLs in 29 strains co-harbored with other β-lactamases using standard microbiological practices and phenotypic procedures. Minimum inhibitory concentrations (MICs) were determined against several antibiotics using Microscan WalkAway plus. OMP analysis was carried out using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ESBL, AmpC, and carbapenemase genes were detected using molecular methods. The microbiological analysis discovered 29 (36.7%) ESBL strains of K. pneumoniae, which showed the co-existence of 7 (24.1%) AmpC β-lactamases and 22 (75.9%) carbapenemases. Porin loss of OmpK35 was observed in 13 (44.8%) and OmpK36 in 8 (27.5%) K. pneumoniae strains. The strains were significantly associated with the intensive care unit (ICU) (p = 0.006) and urinary sources (p = 0.004). The most commonly detected gene variants in each β-lactamase class included 16 (55.2%) bla_CTX-M−1, 7 (100%) bla_CYM-2, 11 (50%) bla_NDM-1, and integron-1 was detected in 21/29 (72.4%) strains. MICs of cephalosporin, fluoroquinolone, carbapenem, aminoglycoside, and β-lactam combinations demonstrated a high number of XDR strains. Tigecycline (2 µg/mL MIC₅₀ and >32 µg/mL MIC₉₀) and colistin (1 µg/mL MIC₅₀ and 8 µg/mL MIC₉₀) presented lower resistance. ESBL K. pneumoniae strains with OmpK35 and OmpK36 porin loss demonstrate conglomerate resistance mechanisms with AmpC and carbapenemases, leading to emerging XDR and pan drug resistance.     

Quantifying Nonspecific TEM β-Lactamase (blaTEM) Genes in a Wastewater Stream

To control the antibiotic resistance epidemic, it is necessary to understand the distribution of genetic material encoding antibiotic resistance in the environment and how anthropogenic inputs, such as wastewater, affect this distribution. Approximately two-thirds of antibiotics administered to humans are β-lactams, for which the predominant bacterial resistance mechanism is hydrolysis by β-lactamases. Of the β-lactamases, the TEM family is of overriding significance with regard to diversity, prevalence, and distribution. This paper describes the design of DNA probes universal for all known TEM β-lactamase genes and the application of a quantitative PCR assay (also known as Taqman) to quantify these genes in environmental samples. The primer set was used to study whether sewage, both treated and untreated, contributes to the spread of these genes in receiving waters. It was found that while modern sewage treatment technologies reduce the concentrations of these antibiotic resistance genes, the ratio of bla_TEM genes to 16S rRNA genes increases with treatment, suggesting that bacteria harboring bla_TEM are more likely to survive the treatment process. Thus, β-lactamase genes are being introduced into the environment in significantly higher concentrations than occur naturally, creating reservoirs of increased resistance potential.     

Sub-Inhibitory Cefsulodin Sensitization of E. coli to β-lactams Is Mediated by PBP1b Inhibition

The combination of antibiotics is one of the strategies to combat drug-resistant bacteria, though only a handful of such combinations are in use, such as the β-lactam combinations. In the present study, the efficacy of a specific sub-inhibitory concentration of cefsulodin with other β-lactams was evaluated against a range of Gram-negative clinical isolates. This approach increased the sensitivity of the isolates, regardless of the β-lactamase production. The preferred target and mechanism of action of cefsulodin were identified in laboratory strains of Escherichia coli, by examining the effects of deleting the penicillin-binding protein (PBP) 1a and 1b encoding genes individually. Deletion of PBP1b was involved in sensitizing the bacteria to β-lactam agents, irrespective of its O-antigen status. Moreover, the use of a sub-inhibitory concentration of cefsulodin in combination with a β-lactam exerted an effect similar to that one obtained for PBP1b gene deletion. We conclude that the identified β-lactam/cefsulodin combination works by inhibiting PBP1b (at least partially) despite the involvement of β-lactamases, and therefore could be extended to a broad range of Gram-negative pathogens.     

Properties of beta-lactamases produced by three species of mycobacteria

1. Mycobacterium smegmatis (N.C.T.C. 8158), M. fortuitum and M. phlei (MPI) produce a constitutive β-lactamase that has penicillinase and cephalosporinase activity. 2. The β-lactamases of these three species of acid-fast bacteria were mainly cell-bound, only small amounts of activity being liberated into the extracellular fluid. The total β-lactamase activity of these mycobacteria was much lower than that of certain Gram-positive organisms, but comparable with that reported for species of Gram-negative bacteria. 3. The β-lactamases of intact cells of the mycobacteria were not freely accessible to any of the substrates tested, but the apparent crypticity factor to benzylpenicillin was greater than that to cephaloridine and cephalosporin C. 4. Attempts to induce β-lactamase activity in M. smegmatis and M. phlei failed even with high concentrations of inducer. 5. The β-lactamases obtained from the three species of mycobacteria showed different substrate specificities, including different relative activities as cephalosporinases and penicillinases respectively. 6. Certain derivatives of 6-aminopenicillanic acid and 7-aminocephalosporanic acid were found to be resistant to hydrolysis by β-lactamases of M. smegmatis and M. fortuitum. 7. The β-lactamase of M. smegmatis was competitively inhibited by a number of β-lactamase-resistant derivatives of 6-aminopenicillanic acid, but not by similar derivatives of 7-aminocephalosporanic acid.     

Carbapenem Non-Susceptible Enterobacteriaceae in Quebec,Canada: Results of a Laboratory Surveillance Program (2010–2012)

The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) represent a major public health concern because these bacteria are usually extensively resistant to most antibiotics. In order to evaluate their dissemination in Quebec, a surveillance program was introduced in 2010. We report the molecular and epidemiological profiles of CPE isolates collected. Between August 2010 and December 2012, a total of 742 non-duplicate isolates non-susceptible to carbapenems were analysed. AmpC β-lactamase and metallo-β-lactamase production were detected by Etest and carbapenemase production by the modified Hodge test (MHT). Antibiotic susceptibility profiles were determined using broth microdilution or Etest. Clonality of Klebsiella pneumoniae carbapenemase (KPC) strains was analyzed by pulsed-field gel electrophoresis (PFGE). The presence of genes encoding carbapenemases as well as other β-lactamases was detected using PCR. Of the 742 isolates tested, 169 (22.8%) were CPE. Of these 169 isolates, 151 (89.3%) harboured a bla _KPC gene while the remaining isolates carried bla _SME (n = 9), bla _OXA-48 (n = 5), bla _NDM (n = 3), and bla _NMC (n = 1) genes. Among the 93 KPC strains presenting with a unique pattern (unique PFGE pattern and/or unique antibiotics susceptibility profile), 99% were resistant to ertapenem, 95% to imipenem, 87% to meropenem, 97% to aztreonam, 31% to colistin and 2% to tigecycline. In 19 patients, 2 to 5 KPC strains from different species or with a different PFGE pattern were isolated. CPE strains were present in the province of Quebec with the majority of strains harbouring KPC. Alternately, SME, OXA-48 and NMC containing strains were rarely found.     

Heavy-Metal and Antibiotic Resistance in the Bacterial Flora of Sediments of New York Bight

The New York Bight extends seaward some 80 to 100 miles (ca. 129 to 161 km) from the Long Island and New Jersey shorelines to the edge of the continental shelf. Over 14 × 10⁶ m³ of sewage sludge, dredge spoils, acid wastes, and cellar dirt are discharged into this area each year. Large populations of Bacillus sp. resistant to 20 μg of mercury per ml were observed in Bight sediments contaminated by these wastes. Resistant Bacillus populations were much greater in sediments containing high concentrations of Hg and other heavy metals than in sediments from areas further offshore where dumping has never been practiced and where heavy-metal concentrations were found to be low. Ampicillin resistance due mainly to β-lactamase production was significantly (P < 0.001) more frequent in Bacillus strains from sediments near the sewage sludge dump site than in similar Bacillus populations from control sediments. Bacillus strains with combined ampicillin and Hg resistances were almost six times as frequent at the sludge dump site as in control sediments. This observation suggests that genes for Hg resistance and β-lactamase production are simultaneously selected for in Bacillus and that heavy-metal contamination of an ecosystem can result in a selection pressure for antibiotic resistance in bacteria in that system. Also, Hg resistance was frequently linked with other heavy-metal resistances and, in a substantial proportion of Bacillus strains, involved reduction to volatile metallic Hg (Hg°).     

Combinatorial active-site variants confer sustained clavulanate resistance in BlaC β-lactamase from Mycobacterium tuberculosis

Bacterial resistance to β-lactam antibiotics is a global issue threatening the success of infectious disease treatments worldwide. Mycobacterium tuberculosis has been particularly resilient to β-lactam treatment, primarily due to the chromosomally encoded BlaC β-lactamase, a broad-spectrum hydrolase that renders ineffective the vast majority of relevant β-lactam compounds currently in use. Recent laboratory and clinical studies have nevertheless shown that specific β-lactam–BlaC inhibitor combinations can be used to inhibit the growth of extensively drug-resistant strains of M. tuberculosis, effectively offering new tools for combined treatment regimens against resistant strains. In the present work, we performed combinatorial active-site replacements in BlaC to demonstrate that specific inhibitor-resistant (IRT) substitutions at positions 69, 130, 220, and/or 234 can act synergistically to yield active-site variants with several thousand fold greater in vitro resistance to clavulanate, the most common clinical β-lactamase inhibitor. While most single and double variants remain sensitive to clavulanate, double mutants R220S-K234R and S130G-K234R are substantially less affected by time-dependent clavulanate inactivation, showing residual β-lactam hydrolytic activities of 46% and 83% after 24 h incubation with a clinically relevant inhibitor concentration (5 μg/ml, 25 µM). These results demonstrate that active-site alterations in BlaC yield resistant variants that remain active and stable over prolonged bacterial generation times compatible with mycobacterial proliferation. These results also emphasize the formidable adaptive potential of inhibitor-resistant substitutions in β-lactamases, potentially casting a shadow on specific β-lactam–BlaC inhibitor combination treatments against M. tuberculosis.     

Genetic Diversity in the Lactose Operons of Lactobacillus helveticus Strains and Its Relationship to the Role of These Strains as Commercial Starter Cultures

Two novel insertion sequence elements, ISLhe1 and ISLhe15, were located upstream of the genes encoding the β-galactosidase enzyme in Lactobacillus helveticus commercial starter strains. Strains with the IS982 family element, ISLhe1, demonstrated reduced β-galactosidase activity compared to the L. helveticus type strain, whereas strains with the ISLhe15 element expressed β-galactosidase in the absence of lactose.     

Extended-Spectrum β-Lactamase CTX-M-1 in Escherichia coli Isolates from Healthy Poultry in France

Genes encoding extended-spectrum β-lactamase CTX-M-1 were detected in 12 Escherichia coli isolates recovered over a 7-month period from the ceca of healthy poultry in seven districts in France in 2005. Eleven of those strains were not clonally related and had a bla_CTX-M-1 gene located on transferable plasmids of different sizes and structures.     

Heterologous Expression of Lactose- and Galactose-Utilizing Pathways from Lactic Acid Bacteria in Corynebacterium glutamicum for Production of Lysine in Whey

The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and β-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited β-galactosidase activity in excess of 1,000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and β-galactosidase genes exhibited β-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability.     

β-Lactam Effects on Mixed Cultures of Common Respiratory Isolates as an Approach to Treatment Effects on Nasopharyngeal Bacterial Population Dynamics

Background

Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae are bacteria present in the nasopharynx as part of normal flora. The ecological equilibrium in the nasopharynx can be disrupted by the presence of antibiotics.

Methodology/Principal Findings

A computerized two-compartment pharmacodynamic model was used to explore β-lactam effects on the evolution over time of a bacterial load containing common pharyngeal isolates by simulating free serum concentrations obtained with amoxicillin (AMX) 875 mg tid, amoxicillin/clavulanic acid (AMC) 875/125 mg tid and cefditoren (CDN) 400 mg bid regimens over 24 h. Strains and MICs (µg/ml) of AMX, AMC and CDN were: S. pyogenes (0.03, 0.03 and 0.015), S. pneumoniae (2, 2 and 0.25), a β-lactamase positive H. influenzae (BL⁺; >16, 2 and 0.06) and a β-lactamase positive AMC-resistant H. influenzae (BLPACR, >16, 8 and 0.06). Mixture of identical 1∶1∶1∶1 volumes of each bacterial suspension were prepared yielding an inocula of ≈4×10⁶ cfu/ml. Antibiotic concentrations were measured both in bacterial and in bacteria-free antibiotic simulations. β-lactamase production decreased AMX concentrations and fT_>MIC against S. pneumoniae (from 43.2% to 17.7%) or S. pyogenes (from 99.9% to 24.9%), and eradication was precluded. The presence of clavulanic acid countered this effect of co-pathogenicity, and S. pyogenes (but not BL⁺ and S. pneumoniae) was eradicated. Resistance of CDN to TEM β-lactamase avoided this co-pathogenicity effect, and CDN eradicated S. pyogenes and H. influenzae strains (fT_>MIC >58%), and reduced in 94% S. pneumoniae counts (fT_>MIC ≈25%).

Conclusions/Significance

Co-pathogenicity seems to be gradual since clavulanic acid countered this effect for strains very susceptible to AMX as S. pyogenes but not for strains with AMX MIC values in the limit of susceptibility as S. pneumoniae. There is a potential therapeutic advantage for β-lactamase resistant cephalosporins with high activity against streptococci.     

Genome Sequence of Stenotrophomonas maltophilia S028, an Isolate Harboring the AmpR-L2 Resistance Module

Multidrug-resistant Stenotrophomonas maltophilia has emerged as an important cause of nosocomial infections, which is attributable mainly to the production of diverse β-lactamases by S. maltophilia. The L2 β-lactamase mediated by the AmpR-L2 module is the most represented lactamase. Here, we announce the genome sequence of S028, an isolate harboring the AmpR-L2 module.     

The Tandem Repeats Enabling Reversible Switching between the Two Phases of β-Lactamase Substrate Spectrum

Expansion or shrinkage of existing tandem repeats (TRs) associated with various biological processes has been actively studied in both prokaryotic and eukaryotic genomes, while their origin and biological implications remain mostly unknown. Here we describe various duplications (de novo TRs) that occurred in the coding region of a β-lactamase gene, where a conserved structure called the omega loop is encoded. These duplications that occurred under selection using ceftazidime conferred substrate spectrum extension to include the antibiotic. Under selective pressure with one of the original substrates (amoxicillin), a high level of reversion occurred in the mutant β-lactamase genes completing a cycle back to the original substrate spectrum. The de novo TRs coupled with reversion makes a genetic toggling mechanism enabling reversible switching between the two phases of the substrate spectrum of β-lactamases. This toggle exemplifies the effective adaptation of de novo TRs for enhanced bacterial survival. We found pairs of direct repeats that mediated the DNA duplication (TR formation). In addition, we found different duos of sequences that mediated the DNA duplication. These novel elements—that we named SCSs (same-strand complementary sequences)—were also found associated with β-lactamase TR mutations from clinical isolates. Both direct repeats and SCSs had a high correlation with TRs in diverse bacterial genomes throughout the major phylogenetic lineages, suggesting that they comprise a fundamental mechanism shaping the bacterial evolution.     

Characterization of Two New CTX-M-25-Group Extended-Spectrum β-Lactamase Variants Identified in Escherichia coli Isolates from Israel

Objectives

We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains.

Methods

ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla _CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla _CTX-M-94 and bla _CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla _CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants.

Results

The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla _CTX-M-94 and bla _CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons.

Conclusions

This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.     

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.