Studies on the mode of action and beta-lactamase inhibitory properties of novel oxapenem compounds

Four novel oxapenem compounds were evaluated for their ß-lactamase inhibitory and antibacterial properties. Two (AM-112 and AM-113) displayed intrinsic antibacterial activity with MICs of between 2 to 16µg/ml and 0.5-2µg/ml against Escherichia coli and methicillin-sensitive and -resistant Staphylococcus aureus, respectively. The isomers of these compounds, AM-115 and AM-114 did not display significant antibacterial activity. Combination of the oxapenems with ceftazidime afforded protection against ß-lactamase-producing strains, including hyperproducers of class C enzymes and extended-spectrum ß-lactamase enzymes. A fixed 4µg/ml concentration of AM-112 protected a panel of eight cephalosporins against hydrolysis by class A and class C ß-lactamase producers. In vivo studies confirmed the protective effect of AM-112 for ceftazidime against ß-lactamase producing S. aureus, Enterobacter cloacae and E. coli strains in a murine intraperitoneal infection model. Each of the oxapenems inhibited class A, class C and class D ß-lactamases isolated from whole cells and purified by isoelectric focusing. AM-114 and AM-115 were as effective as clavulanic acid against class A enzymes. AM-112 and AM-113 were less potent against these enzymes. Class C and class D enzymes proved very susceptible to inhibition by the oxapenems. Molecular modelling of the oxapenems in the active site of the class A. TEM-1 and class C P99 enzymes identified a number of potential sites of interaction. The modelling suggested that Ser-130 in TEM-1 and Tyr-150 in P99 were likely candidates for cross-linking of the inhibitor, leading to inhibition of the enzyme. Morphology studies indicated that sub-inhibitory concentrations of the oxapenems caused the formation of round-shaped cells in E. coli DC0, indicating inhibition of penicillin-binding protein 2 (PBP2). The PBP affinity profile of AM-112 was examined in isolated cell membranes of E. coli DC0, S. aureus NCTC 6571, Enterococcus faecalis SFZ and E. faecalis ATCC 29213, in competition with a radiolabelled penicillin. PBP2 was identified as the primary target for AM-112 in E. coli DC0. Studies on S. aureus NCTC 6571 failed to identify a binding target. AM-112 bound to all the PBPs of both E. faecalis strains, and a concentration of 10µg/ml inhibited all the PBPs except PBP3.

Synthesis and evaluation of antibacterial activity of the dual-action agents: beta-lactamase inhibitors with cytotoxic agents or beta-lactam antibiotics

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Characterization of the amp genes involved in the regulation of beta-lactamase expression in Pseudomonas aeruginosa

Antibiotic resistance, production of alginate and virulence factors, and altered host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection. Failure of antibiotic therapy has been attributed to the emergence of P. aeruginosa strains that produce β-lactamase constitutively. In Enterobacteriaceae, β-lactamase induction involves four genes with known functions: ampC, ampR, ampD, and ampG, encoding the enzyme, transcriptional regulator, amidase and permease, respectively. In addition to all these amp genes, P. aeruginosa possesses two ampG paralogs, designated ampG and ampP. In this study, P. aeruginosa ampC, ampR, ampG and ampP were analyzed. Inactivation of ampC in the prototypic PAO1 failed to abolish the β-lactamase activity leading to the discovery of P. aeruginosa oxacillinase PoxB. Cloning and expression of poxB in Escherichia coli confers β-lactam resistance. Both AmpC and PoxB contribute to P. aeruginosa resistance against a wide spectrum of β-lactam antibiotics. The expression of PoxB and AmpC is regulated by a LysR-type transcriptional regulator AmpR that up-regulates AmpC but down-regulates PoxB activities. Analyses of P. aeruginosa ampR mutant demonstrate that AmpR is a global regulator that modulates the expressions of Las and Rhl quorum sensing (QS) systems, and the production of pyocyanin, LasA protease and LasB elastase. Introduction of the ampR mutation into an alginate-producing strain reveals the presence of a complex co-regulatory network between antibiotic resistance, QS alginate and other virulence factor production. Using phoA and lacZ protein fusion analyses, AmpR, AmpG and AmpP were localized to the inner membrane with one, 16 and 10 transmembrane helices, respectively. AmpR has a cytoplasmic DNA-binding and a periplasmic substrate binding domains. AmpG and AmpP are essential for the maximal expression of β-lactamase. Analysis of the murein breakdown products suggests that AmpG exports UDP-N-acetylmuramyl-L-alanine-γ-D-glutamate-meso-diaminopimelic acid-D-alanine-D-alanine (UDP-MurNAc-pentapeptide), the corepressor of AmpR, whereas AmpP imports N-acetylglucosaminyl-beta-1,4-anhydro-N-acetylmuramic acid-Ala-γ-D-Glu-meso-diaminopimelic acid (GlcNAc-anhMurNAc-tripeptide) and GlcNAc-anhMurNAc-pentapeptide, the co-inducers of AmpR. This study reveals a complex interaction between the Amp proteins and murein breakdown products involved in P. aeruginosa β-lactamase induction. In summary, this dissertation takes us a little closer to understanding the P. aeruginosa complex co-regulatory mechanism in the development of β-lactam resistance and establishment of chronic infection. ^

Dissemination of bla(IMP-1)-carrying integron In86 among Klebsiella pneumoniae isolates harboring a new trimethoprim resistance gene dfr23

The genetic context of the bla(IMP-1) gene was evaluated in 9 Klebsiella pneumoniae isolates recovered from 2 hospitals in Sao Paulo, Brazil. All isolates harbored a copy of In86 carrying bla(Imp-1), aac(6`)-31, and aadAl. Eight strains from the same hospital also carried another class I integron harboring a new trimethoprim resistance gene (dfr23) that was chromosomally embedded. In86 was likely to be in a 30-kb nontransferable plasmid and was flanked upstream by a sequence identical to one identified in an IMP-1-producing Pseudomonas putida isolate. The bla(IMP-1)-carrying integron In86 was recently reported from nonfermentative bacilli isolated in Sao Paulo. These isolates appear to be the Source of this integron now acquired by K. pneumoniae strains from different hospitals in the same city. Metallo-beta-lactamase production is still rare among Enterobacteriaceae isolates in Brazil, but the acquisition of genetic structures carrying these mobile resistance determinants is worrisome and could lead to an increase in the prevalence of these phenotypes of resistance. (C) 2008 Elsevier Inc. All rights reserved.

The characteristics of NDM-producing Klebsiella pneumoniae from Canada

After recent hospitalization in India (New Delhi and Mumbai), 2 patients, on their return to Canada, presented with lower urinary tract infections due to multiresistant Klebsiella pneumoniae that produced New Delhi metall-beta-lactamase and CTX-M-15. The organisms belonged to clones ST147 and ST340, and were positive for aac(6`)-Ib-cr, as well as for the ccdAB and vagCD addiction systems. The bla(NDM) plasmid was located on the IncFIIA and IncA/C replicon groups of plasmids. Clones ST147 and ST340 are also responsible for harbouring bla(KPC), and it is possible that they played an important role in the intercontinental spread of antimicrobial resistance. (C) 2011 Elsevier Inc. All rights reserved.

The Challenges of Antimicrobial Resistance in Brazil

Brazil is a country with continental proportions with high geographic and economic diversity. Despite its medical centers of excellence, antimicrobial resistance poses a major therapeutic challenge. Rates of methicillin-resistant Staphylococcus aureus are up to 60% and are related to an endemic Brazilian clone. Local resistance to vancomycin in Enterococci was first related to Enterococcus faecalis, which differs from European and American epidemiology. Also, local Klebsiella pneumoniae and Escherichia coli isolates producing extended-spectrum beta-lactamases have a much higher prevalence (40%-50% and 10%-18%, respectively). Carbapenem resistance among the enterobacteriaceae group is becoming a major problem, and K. pneumoniae carbapenemase isolates have been reported in different states. Among nonfermenters, carbapenem resistance is strongly related to SPM-1 (Pseudomonasaeruginosa) and OXA-23 (Acinetobacter baumannii complex) enzymes, and a colistin-only susceptible phenotype has also emerged in these isolates, which is worrisome. Local actions without loosing the global resistance perspective will demand multidisciplinary actions, new policies, and political engagement.

Detection of P. aeruginosa harboring bla(CTX-M-2), bla(GES-1) and bla(GES-5), bla(IMP-1) and bla(SPM-1) causing infections in Brazilian tertiary-care hospital

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Penicillinase-based amperometric biosensor for penicillin G

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

High genetic diversity among Pseudomonas aeruginosa and Acinetobacter spp. isolated in a public hospital in Brazil

In Brazil and other regions of the world, Pseudomonas aeruginosa and Acinetobacter spp. have emerged as important agents of nosocomial infection and are commonly involved in outbreaks. The main objective of the present study was to evaluate the genetic relationship among P. aeruginosa and Acinetobacter spp. isolated from patients in a public university hospital in northwestern Parana, Brazil, and report their antimicrobial resistance profile. A total of 75 P. aeruginosa and 94 Acinetobacter spp. isolates were phenotypically identified and tested for antibiotic susceptibility using automated methodology. Polymyxin B was tested by disk diffusion for P. aeruginosa. Metallo-beta-lactamase (MBL) was detected using a disk approximation test. Genotyping was performed using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Approximately 55% of the P. aeruginosa isolates and 92% of the Acinetobacter spp. isolates were multiresistant, but none were MBL-producers. ERIC-PCR revealed the presence of small clusters of carbapenem-resistant Acinetobacter spp., most likely OXA-type carbapenemase producers. Furthermore, high genetic diversity in P. aeruginosa and Acinetobacter spp. clinical isolates was observed, suggesting that cross-transmission is not very frequent in the studied hospital.

The characteristics of VIM-1-producing Klebsiella pneumoniae from South Africa

A study was designed to characterize a carbapenem-resistant Klebsiella pneumoniae (KPSA01) isolated from a patient in Gauteng, South Africa without recent travel outside South Africa. Molecular characterization was done using isoelectric focusing, polymerase chain reaction and sequencing for bla(VIM), bla(IMP), bla(NDM), bla(CTX-Ms), bla(OXAs), bla(TEMs), and bla(SHV), plasmid-mediated quinolone resistance determinants, multilocus sequencing typing, plasmid replicon typing, and addiction factors. KPSA01 produced VIM-1 and belonged to the newly described sequence type ST569. The plasmid that harboured bla(VIM) typed within the narrow host range IncF replicon group, contained the aadA1 gene cassette, and tested positive for the vagCD and ccdAB addiction systems. This is the first report of VIM-1-producing K. pneumoniae outside Europe. It is important that surveillance studies be undertaken in Africa to determine if VIM-1-producing K. pneumoniae are present in significant numbers.

Determinants of beta-lactam resistance in meningitis-causing Enterobacteriaceae in Brazil

This study analyzed resistance determinants in extended-spectrum beta-lactamase (ESBL)-producing enterobacteria and the epidemiology of 11 Escherichia coli isolates obtained from meningitis patients in a region of Brazil from 2000 to 2005. ESBL-encoding genes and their genetic environment were investigated by PCR and sequencing. The gene bla(CTX-M-2) was identified in 3 different enterobacteria (E. coli. Serratia marcescens, and Proteus mirabilis) downstream of the insertion sequence ISCR1 (localized in class 1 integrons), hut not as part of the resistance cassettes region. Multi locus sequence typing (MLST) was used to investigate genetic relationships between the 11 E. coil isolates in this study and strains associated with meningitis in the E. coil MLST database. MLST analysis indicated high genetic diversity among isolates, and no significant genetic relationship was identified with meningitis-causing E. coil in the database. The results in this report reinforce the need to be attentive to meningitis suspected to be due to ESBL-producing enterobacterial isolates, especially where ESBL epidemiology is well known.

Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia

Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla(SHV-2a) gene and seven strains carried the bla(SHV-12) gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla(SHV-11) gene and one strain carried the non-ESBL bla(SHV-1) gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla(SHV-2a) or bla(SHV12) gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed first-nucleotide change, involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla(SHV-2a) from strains carrying bla(SHV-12). In addition, this method was used to demonstrated an association between the relative copy numbers of bla(SHV) genes in individual strains and the levels of antibiotic resistance.

Risk factors for and mortality of extended-spectrum-β-lactamase-producing Klebsiella pneumoniae and Escherichia coli nosocomial bloodstream infections

A case-control study, involving patients with positive blood cultures for Klebsiella pneumoniae (KP) or Escherichia coli (EC) EC and controls with positive blood cultures for non-ESBL-KP or EC, was performed to assess risk factors for extended-spectrum-β-lactamase (ESBL) production from nosocomial bloodstream infections (BSIs). Mortality among patients with BSIs was also assessed. The study included 145 patients (81, 59.5% with K. pneumoniae and 64, 44.1% with E. coli BSI); 51 (35.2%) isolates were ESBL producers and 94 (64.8%) nonproducers. Forty-five (55.6%) K. pneumoniae isolates were ESBL producers, while only six (9.4%) E. coli isolates produced the enzyme. Multivariate analysis showed that recent exposure to piperacillin-tazobactam (adjusted Odds Ratio [aOR] 6.2; 95%CI 1.1-34.7) was a risk factor for ESBL BSI. K. pneumoniae was significantly more likely to be an ESBL-producing isolate than E. coli (aOR 6.7; 95%CI 2.3-20.2). No cephalosporin class was independently associated with ESBLs BSI; however, in a secondary model considering all oxymino-cephalosporins as a single variable, a significant association was demonstrated (aOR 3.7; 95%CI 1.3-10.8). Overall 60-day mortality was significantly higher among ESBL-producing organisms. The finding that piperacillin-tazobactam use is a risk factor for ESBL-production in KP or EC BSIs requires attention, since this drug can be recommended to limit the use of third-generation cephalosporins.