Active sites of beta-lactamases. The chromosomal beta-lactamases of Pseudomonas aeruginosa and Escherichia coli.
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ABSTRACT:
SUBMITTER: Knott-Hunziker V
PROVIDER: S-EPMC1163689 | biostudies-other | 1982 Mar
REPOSITORIES: biostudies-other
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Project description:Metallo-β-lactamases (MBLs) have rapidly disseminated worldwide among clinically important Gram-negative bacteria and have challenged the therapeutic use of β-lactam antibiotics, particularly carbapenems. The bla(GIM-1) gene, encoding one such enzyme, was first discovered in a Pseudomonas aeruginosa isolate from 2002 and has more recently been reported in Enterobacteriaceae. Here, we present crystal structures of GIM-1 in the apo-zinc (metal-free), mono-zinc (where Cys221 was found to be oxidized), and di-zinc forms, providing nine independently refined views of the enzyme. GIM-1 is distinguished from related MBLs in possessing a narrower active-site groove defined by aromatic side chains (Trp228 and Tyr233) at positions normally occupied by hydrophilic residues in other MBLs. Our structures reveal considerable flexibility in two loops (loop 1, residues 60 to 66; loop 2, residues 223 to 242) adjacent to the active site, with open and closed conformations defined by alternative hydrogen-bonding patterns involving Trp228. We suggest that this capacity for rearrangement permits GIM-1 to hydrolyze a wide range of β-lactams in spite of possessing a more constrained active site. Our results highlight the structural diversity within the MBL enzyme family.
| S-EPMC3553699 | biostudies-literature
Project description:BackgroundCarbapenems are important drugs used for the treatment of Pseudomonas aeruginosa infections, however metallo-β-lactamases (MBL) are able to efficiently hydrolyze these classes of drugs. Immediate detection of the MBL-producing P. aeruginosa is necessary in order to accurately treat infections caused by this organism.ObjectivesTo determine the prevalence of MBL producing P. aeruginosa in burn and non-burn patients by two phenotypic tests and polymerase chain reaction (PCR) and to compare phenotypic tests with PCR.Materials and methodsA total of 223 non-duplicate strains of P. aeruginosa were collected from three teaching hospitals of Ahvaz, Iran. Antimicrobial susceptibility and minimum inhibitory concentrations (MICs) of carbapenems (imipenem, meropenem, doripenem and ertapenem) were determined by the Kirby-Bauer and E-test methods. Combined disk (CD) test, MBL E-test and PCR were performed for carbapenem-resistant P. aeruginosa isolates.ResultsAmongst all the P. aeruginosa isolates, 58.7% were resistant to imipenem while 31.8%, 13.5% and 74.4% were resistant to meropenem, doripenem and ertapenem, respectively. Amongst all the P. aeruginosa isolates, 44.4% were multidrug resistant and 13.45% were resistant to all of the carbapenems. The CD test with doripenem disk / 750 μg ethylene diamine tetra acetic acid (EDTA) had the highest efficiency compared to the other phenotypic tests. bla IMP and bla VIM genes were detected in 11.7% and 0.4% of isolates, respectively. bla SPM and bla NDM genes were not observed.ConclusionsEpidemiological and regional evaluation of MBL-producing P. aeruginosa through simple and inexpensive methods should be considered for effective treatment of carbapenem-resistant P. aeruginosa infections.
| S-EPMC4332233 | biostudies-literature
Project description:With the occurrence of extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa being increasingly reported worldwide, there is a need for a reliable test to detect ESBLs in clinical isolates of P. aeruginosa. In our study, a total of 75 clinical isolates of P. aeruginosa were studied. Nitrocefin tests were performed to detect the beta-lactamase enzyme; isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to further characterize the contained ESBLs. Various ESBL-screening methods were designed to compare the reliabilities of detecting ESBLs in clinical isolates of P. aeruginosa whose beta-lactamases were well characterized. Thirty-four of 36 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBLs. bla(VEB-3) was the most prevalent ESBL gene in P. aeruginosa in our study. Among the total of 34 isolates that were considered ESBL producers, 20 strains were positive using conventional combined disk tests and 10 strains were positive using a conventional double-disk synergy test (DDST) with amoxicillin-clavulanate, expanded-spectrum cephalosporins, aztreonam, and cefepime. Modifications of the combined disk test and DDST, which consisted of shorter distances between disks (20 mm instead of 30 mm) and the use of three different plates that contained cloxacillin (200 microg/ml) alone, Phe-Arg beta-naphthylamide dihydrochloride (MC-207,110; 20 microg/ml) alone, and both cloxacillin (200 microg/ml) and MC-207,110 (20 microg/ml) increased the sensitivity of the tests to 78.8%, 91.18%, 85.29%, and 97.06%.
| S-EPMC1563526 | biostudies-literature
Project description:ObjectivesThe siderophore cephalosporin cefiderocol possesses in vitro activity against MDR Gram-negative bacteria. The stability of cefiderocol against serine- and metallo-type carbapenemases has been reported previously, but little is known about how cefiderocol interacts with chromosomal AmpC ?-lactamases. We investigated a number of features of cefiderocol, namely antibacterial activity against AmpC overproducers, stability against AmpC ?-lactamases and propensity for AmpC induction using Pseudomonas aeruginosa and Enterobacter cloacae.MethodsMICs were determined by broth microdilution according to CLSI guidelines. The MIC of cefiderocol was determined in iron-depleted CAMHB. Hydrolysis of the antibiotics was determined by monitoring the changes in the absorbance in the presence of AmpC ?-lactamase, and AmpC induction was evaluated by double disc diffusion and nitrocefin degradation assays.ResultsThe MICs of ceftazidime and cefepime for PAO1 increased 4- to 16-fold with inactivation of either ampD or dacB, whereas cefiderocol MICs were little affected by these inactivations (<2-fold increase). Cefiderocol has 17- and 740-fold lower affinity (higher Ki) to AmpCs of P. aeruginosa SR24-12 and E. cloacae P99, respectively, compared with ceftazidime. Both disc diffusion and nitrocefin degradation assays indicated that cefiderocol did not induce AmpC ?-lactamases of P. aeruginosa PAO1 and ATCC 27853 and E. cloacae ATCC 13047, whereas imipenem did.ConclusionsCefiderocol showed in vitro activity against the AmpC-overproducing strains, low affinity for chromosomal AmpC ?-lactamases, and a low propensity of temporal induction of AmpC ?-lactamases of P. aeruginosa and E. cloacae. These features relating to chromosomal AmpC could explain the potent antibacterial activity of cefiderocol against drug-resistant strains producing AmpC ?-lactamases.
| S-EPMC6198743 | biostudies-literature
Project description:A nationwide study aimed to identify the extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases (MBLs), and extended-spectrum oxacillinases (ES-OXAs) in a French collection of 140 clinical Pseudomonas aeruginosa isolates highly resistant to ceftazidime. Six ESBLs (PER-1, n=3; SHV-2a, n=2; VEB-1a, n=1), four MBLs (VIM-2, n=3; IMP-18, n=1), and five ES-OXAs (OXA-19, n=4; OXA-28, n=1) were identified in 13 isolates (9.3% of the collection). The prevalence of these enzymes is still low in French clinical P. aeruginosa isolates but deserves to be closely monitored.
| S-EPMC2916347 | biostudies-literature
Project description:Biofilms have been implicated as an important reservoir for pathogens and commensal enteric bacteria such as Escherichia coli in natural and engineered water systems. However, the processes that regulate the survival of E. coli in aquatic biofilms have not been thoroughly studied. We examined the effects of hydrodynamic shear and nutrient concentrations on E. coli colonization of pre-established Pseudomonas aeruginosa biofilms, co-inoculation of E. coli and P. aeruginosa biofilms, and P. aeruginosa colonization of pre-established E. coli biofilms. In nutritionally-limited R2A medium, E. coli dominated biofilms when co-inoculated with P. aeruginosa, and successfully colonized and overgrew pre-established P. aeruginosa biofilms. In more enriched media, P. aeruginosa formed larger clusters, but E. coli still extensively overgrew and colonized the interior of P. aeruginosa clusters. In mono-culture, E. coli formed sparse and discontinuous biofilms. After P. aeruginosa was introduced to these biofilms, E. coli growth increased substantially, resulting in patterns of biofilm colonization similar to those observed under other sequences of organism introduction, i.e., E. coli overgrew P. aeruginosa and colonized the interior of P. aeruginosa clusters. These results demonstrate that E. coli not only persists in aquatic biofilms under depleted nutritional conditions, but interactions with P. aeruginosa can greatly increase E. coli growth in biofilms under these experimental conditions.
| S-EPMC4157881 | biostudies-literature
Project description:The effect of antibiotics on horizontal gene transfer (HGT) is controversial, and the underlying mechanism remains poorly understood. Here, using Escherichia coli SM10λπ as the donor strain, which carries a chromosomally integrated RP4 plasmid, we investigated the effect of antibiotics on conjugational transfer of a mobilizable gentamicin (Gm) resistance plasmid. The results showed that an exposure to gentamicin that restricted the survival of recipient cells significantly enhanced SM10λπ-Pseudomonas aeruginosa PAO1 conjugation, which was attenuated by a deficiency of lasI-rhlI, genes associated with the generation of the quorum sensing signals N-acyl homoserine lactones (AHLs) in PAO1, or the deletion of the AHL receptor SdiA in SM10λπ. Subsequent mechanistic investigations revealed that a treatment with Gm repressed the mRNA expression of lasI and rhlI in PAO1 and upregulated traI expression in SM10λπ. Moreover, PAO1 treated with other quorum sensing (QS)-inhibiting antibiotics such as azithromycin or chloramphenicol also showed a conjugation-promoting ability. On the other hand, when using non-AHL-producing E. coli strain EC600 as the recipient cells, the promoting effect of Gm on conjugation could not be observed. These data suggest that AHL-SdiA contributes to the effectiveness of antibiotics on plasmid conjugation. Collectively, our findings highlight the HGT-promoting effect of antibiotics and suggest quorum sensing as a promising target for controlling antibiotic resistance dissemination. These findings have implications for assessing the risks of antibiotic use and developing advisable antibiotic treatment protocols.
| S-EPMC5700318 | biostudies-literature
Project description:Acquired Metallo-β-Lactamases (MBLs) are emerging resistance determinants in Pseudomonas aeruginosa and other gram-negative bacteria.Using Combination Disk Diffusion test, it was found that among 83 imipenem non-susceptible P. aeruginosa strains, 48 (57.9%) were MBL producers. PCR and Sequencing methods proved that these isolates were positive for blaIMP-1 genes, whereas none were positive for bla(VIM) genes. The mortality rate due to MBL-producing Pseudomonas infection was 4 (8.3%) among the hospitalized patients. Therefore, identification of drug resistance patterns in P. aeruginosa and detection of MBLs producing isolates are of great importance in the prevention and control of infections.
| S-EPMC3636667 | biostudies-literature
Project description:Escherichia coli MutS, MutL and MutH proteins act sequentially in the MMRS (mismatch repair system). MutH directs the repair system to the newly synthesized strand due to its transient lack of Dam (DNA-adenine methylase) methylation. Although Pseudomonas aeruginosa does not have the corresponding E. coli MutH and Dam homologues, and consequently the MMRS seems to work differently, we show that the mutL gene from P. aeruginosa is capable of complementing a MutL-deficient strain of E. coli. MutL from P. aeruginosa has conserved 21 out of the 22 amino acids known to affect functioning of E. coli MutL. We showed, using protein affinity chromatography, that the C-terminal regions of P. aeruginosa and E. coli MutL are capable of specifically interacting with E. coli MutH and retaining the E. coli MutH. Although, the amino acid sequences of the C-terminal regions of these two proteins are only 18% identical, they are 88% identical in the predicted secondary structure. Finally, by analysing (E. coli-P. aeruginosa) chimaeric MutL proteins, we show that the N-terminal regions of E. coli and P. aeruginosa MutL proteins function similarly, in vivo and in vitro. These new findings support the hypothesis that a large surface, rather than a single amino acid, constitutes the MutL surface for interaction with MutH, and that the N- and C-terminal regions of MutL are involved in such interactions.
| S-EPMC1183468 | biostudies-other
Project description:BackgroundChromosomally encoded AmpC β-lactamases may be acquired by transmissible plasmids which consequently can disseminate into bacteria lacking or poorly expressing a chromosomal bla AmpC gene. Nowadays, these plasmid-mediated AmpC β-lactamases are found in different bacterial species, namely Enterobacteriaceae, which typically do not express these types of β-lactamase such as Klebsiella spp. or Escherichia coli. This study was performed to characterize two E. coli isolates collected in two different Portuguese hospitals, both carrying a novel CMY-2-type β-lactamase-encoding gene.FindingsBoth isolates, INSRA1169 and INSRA3413, and their respective transformants, were non-susceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime, but susceptible to cefepime and imipenem, and presented evidence of synergy between cloxacilin and cefoxitin and/or ceftazidime. The genetic characterization of both isolates revealed the presence of bla CMY-46 and bla CMY-50 genes, respectively, and the following three resistance-encoding regions: a Citrobacter freundii chromosome-type structure encompassing a blc-sugE-bla CMY-2-type -ampR platform; a sul1-type class 1 integron with two antibiotic resistance gene cassettes (dfrA1 and aadA1); and a truncated mercury resistance operon.ConclusionsThis study describes two new bla CMY-2-type genes in E. coli isolates, located within a C. freundii-derived fragment, which may suggest their mobilization through mobile genetic elements. The presence of the three different resistance regions in these isolates, with diverse genetic determinants of resistance and mobile elements, may further contribute to the emergence and spread of these genes, both at a chromosomal or/and plasmid level.
| S-EPMC4399151 | biostudies-literature