Contribution of extended-spectrum AmpC (ESAC) beta-lactamases to carbapenem resistance in Escherichia coli

ESAC beta-lactamases have increased catalytic efficiencies toward extended-spectrum cephalosporins and to a lesser extent toward imipenem as compared with the wild-type cephalosporinases. We show here that ESAC expression associated with the loss of both OmpC and OmpF porins conferred in Escherichia coli a high level of resistance to ertapenem and reduced the susceptibility to imipenem. On the contrary, ESAC expressed in the OmpC- or OmpF-deficient E. coli strains or narrow-spectrum cephalosporinase expressed in the OmpC-and OmpF-deficient strain do not confer reduced susceptibility to any of the carbapenems. The production of ESAC beta-lactamase in favorable E. coli background may represent an additional mechanism of resistance to ertapenem.     

Insecticidal and Acaricidal Activity of Certain O,O-Dialkyl O-3-Pyridazinyl Phosphorothioates

Forty-three 3-pyridazinyl phosphorothioates were evaluated for insecticidal and acaricidal activities against two-spotted spider mite, turnip aphid, smaller brown planthopper, mosquito larvae and American cockroach. Approximate acute oral toxicity of these compounds in mice was also examined. In general, the toxicity in mice was in parallel with the pesticidal activity, but a few compounds clearly showed a high degree of selectivity between pests and mice. Especially O,O-dimethyl O-(6-cyclohexyloxy-3-pyridazinyl)phosphorothioate showed substantially reduced mammalian toxicity but maintained high insecticidal and acaricidal activity.     

Antiproliferative activity of Humulus lupulus extracts on human hepatoma (Hep3B), colon (HT-29) cancer cells and proteases,tyrosinase, β-lactamase enzyme inhibition studies

The aims of this study were to examine the antiproliferation of Humulus lupulus extracts on human hepatoma carcinoma (Hep3B) and human colon carcinoma (HT-29) cell lines along with enzyme inhibitory effects of the crude extracts. Potential cell cytotoxicity of six different H. lupulus extracts were assayed on various cancer cells using MTT assay at 24, 48 and 72?h intervals. Methanol-1 extract has inhibited the cell proliferation with doses of 0.6–1?mg/mL in a time dependent (48 and 72 hours) manner in Hep3B cells with 70% inhibition, while inhibitory effect was not seen in colon cancer cells. Acetone extract has increased the cell proliferation at low doses of 0.1?mg/mL for 72?h in Hep3B cells and 0.1–0.2?mg/mL for 48 and 72?h in HT29 cells. The inhibitory effects of the extracts were compared by relative maximum activity values (V_max) using proteases such as α-chymotrypsin, trypsin and papain, tyrosinase and β-lactamase (penicillinase).     

Detection By Whole-Genome Sequencing of a Novel Metallo-β-Lactamase Produced By Wautersiella Falsenii Causing Urinary Tract Infection in Tunisia

Wautersiella falsenii is a rarely non-fermenting Gram-negative bacterium and belongs to the Flavobacteriaceae family. This nosocomial pathogen can cause several human infections, especially among immunocompromised patients. Here, we describe the whole genome sequence of a clinical W. falsenii strain isolated from a urine sample of a 35-year-old woman with a urinary tract infection in Tunisia. We investigated its phenotype and genotype.After bacterial identification by the MALDI-TOF method, the whole-genome sequencing of this strain was performed. This isolate was not susceptible to various antibiotics, including β-lactams, aminoglycosides, and quinolones. However, it remains susceptible to imipenem (MIC = 0.25 mg/l), ertapenem (MIC = 0.75 mg/l), and meropenem (MIC = 0.19 mg/l). Interestingly, the E-TEST^® (MP/MPI) showed a reduced MIC of meropenem +/− EDTA (0.064 μg/ml). Besides, the color change from yellow to red in the β CARBA test only after 24 hours of incubation can be interpreted in two ways. On the one hand, as a likely low expression of the gene encoding metallo-β-lactamase. On the other hand, and more likely, it may be a false-positive result because, according to the test manufacturer''s recommendations, the test should be read after 30 minutes. Perhaps, therefore, this gene is not expressed in the tested strain. Moreover, the whole-genome sequence analysis demonstrated the presence of a novel chromosomally located subclass B1 metallo-β-lactamase EBR-like enzyme, sharing 94.92% amino acid identity with a previously described carbapenemase produced by Empedobacter brevis, EBR-1. The results also showed the detection of other antibiotic resistance genes and the absence of plasmids. So far, this study is the first report on the detection of W. falsenii in Tunisia. These findings prove that W. falsenii could be a potential reservoir of antibiotic resistance genes, e.g., β-lactamases. Collaborative efforts and effective hygiene measures should be established to prevent the emergence of this species in our health care settings.     

Slow Protein Dynamics Elicits New Enzymatic Functions by Means of Epistatic Interactions

Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M β-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried β-sheet. The two mutations located in this β-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M β-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.     

Azolylthioacetamides as a potent scaffold for the development of metallo-β-lactamase inhibitors

In an effort to develop new inhibitors of metallo-β-lactamases (MβLs), twenty-eight azolylthioacetamides were synthesized and assayed against MβLs. The obtained benzimidazolyl and benzioxazolyl substituted 1–19 specifically inhibited the enzyme ImiS, and 10 was found to be the most potent inhibitor of ImiS with an IC₅₀ value of 15?nM. The nitrobenzimidazolyl substituted 20–28 specifically inhibited NDM-1, with 27 being the most potent inhibitor with an IC₅₀ value of 170?nM. Further studies with 10, 11, and 27 revealed a mixed inhibition mode with competitive and uncompetitive inhibition constants in a similar range as the IC₅₀ values. These inhibitors resulted in a 2–4-fold decrease in imipenem MIC values using E. coli cells producing ImiS or NDM-1. While the source of uncompetitive (possibly allosteric) inhibition remains unclear, docking studies indicate that 10 and 11 may interact orthosterically with Zn2 in the active site of CphA, while 27 could bridge the two Zn(II) ions in the active site of NDM-1 via its nitro group.     

Study of CTX-M Type of Extended Spectrum β-Lactamase among Nosocomial Isolates of <Emphasis Type=Italic>Escherichia coli</Emphasis> and <Emphasis Type=Italic>Klebsiella pneumoniae</Emphasis> in South India

Data on CTX-M type extended-spectrum β-lactamase (ESBL) produced by Gram-negative bacteria by molecular methods are limited from India. This study was conducted to investigate the prevalence of CTX-M type ESBL producing Escherichia coli and Klebsiella pneumoniae from nosocomial isolates in a tertiary care hospital in southern India. A total of 179 clinical isolates of K. pneumoniae (n = 72) and E. coli (n = 107) were obtained in a period of 3 months and assessed for ESBL production phenotypically. Associated resistance to a panel of antibiotics and Minimum Inhibitory Concentration for 3rd generation cephalosporins was determined. Phenotypically ESBL positive isolates were subjected to PCR for bla _CTX-M gene using two sets of primers for the simultaneous detection of all the five major groups of CTX-M types. All the positive isolates were then subjected to a group specific PCR to detect the prevalent group. Out of 179 isolates, 156 (87.1%) were positive for ESBL phenotypically, which includes 39.2% of K. pneumoniae and 60.8% of E. coli. All of them were examined by PCR using two primers for the presence of bla _CTX-M genes. Among the 156 phenotypic positive isolates, 124 (79.4%) were positive for bla _CTX-M genes, of which 45 (36.2%) were K. pneumoniae, 79 (63.7%) were E. coli. When the 124 positive clinical isolates were further tested with CTX-M group-specific primers, all were positive for the CTX-M-1 group. Our findings document evidence of the high prevalence of multidrug resistant CTX-M group 1 type ESBL among nosocomial isolates in this region. High co-resistance to other non-β-lactam antibiotics is a major challenge for management of ESBL infections. This is alarming and calls for the judicious use of carbapenems, especially in developing countries. This has significant implications for patient management, and indicates the need for increased surveillance and for further molecular characterization of these isolates.     

TRC-1: Emergence of a clavulanic acid-resistant TEM β-lactamase in a clinical strain

A novel TEM-derived plasmid-encoded beta-lactamase, resistant to inhibition by clavulanic acid, has been identified in a clinical strain of Escherichia coli found in Scotland. The beta-lactamase gene was carried on an 81-kb plasmid that conferred no other resistances. The novel enzyme conferred resistance to the amoxycillin/clavulanic acid combination on the host bacterium. The beta-lactamase has a pI of 5.25 and lies between the PSE-4 and SAR-1 beta-lactamases on an isoelectric focusing gel. This beta-lactamase has a Mr value of 25,000, similar to the TEM-1 enzyme and a comparable substrate profile. Its most significant difference is that it is inhibited by clavulanic acid 100-fold less efficiently than the TEM-1 enzyme. The enzyme was confirmed to be derived from the TEM enzymes by probing the plasmid DNA with an intragenic gene probe for TEM-1. This is the first report of a clinical bacterium carrying a TEM-enzyme that confers resistance to clavulanic acid combinations and we have designated the beta-lactamase as TRC-1.     

Molecular characterization of beta-lactamase genes blaA and blaB of Yersinia enterocolitica biovar 1A

The beta-lactamase genes blaA and blaB were detected by PCR amplification in strains of Yersinia enterocolitica biovar 1A isolated from India, Germany, France and the USA. Both genes were detected in all strains. Polymerase chain reaction-restriction fragment length polymorphism revealed genetic heterogeneity in blaA but not in blaB. Cluster analysis of blaA restriction profiles grouped the strains into three groups. The blaA gene of Y. enterocolitica biovar 1A showed a high degree of sequence homology to that of Y. enterocolitica 8081 (biovar 1B) and Y. enterocolitica Y-56 (biovar 4), whereas homology was low with class A beta-lactamase genes of other members of the family Enterobacteriaceae. The pI 8.7 of enzyme Bla-A of Y. enterocolitica biovar 1A was similar to that of biovars 2, 3 and 4. The enzyme Bla-B focused at 6.8 and 7.1, indicating that biovar 1A strains produced a 'B-like' enzyme. This is the first study to have investigated the genetic heterogeneity of the beta-lactamase genes of Y. enterocolitica.