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. ^

Rapid detection of β-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae by use of the new chromogenic βLacta test

The chromogenic βLacta test developed for the rapid detection of β-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae revealed good performance with extended-spectrum β-lactamase (ESBL) producers (97.5% true-positive results). However, false-negative results occurred with chromosomal AmpC hyperproducers and plasmid AmpC producers, whereas uninterpretable results were mostly due to VIM-1 carbapenemase producers and possibly low levels of expressed ESBLs.

Arylsulfatase - producing bacteria in marine sediments

A total of 313 strains of bacteria which hydrolysed tripotassium phenolphthalein disulfate (PDS) were isolated from the sediments of three biotopes, namely, Vellar estuary, backwater and mangrove during the period of investigation. They were identified to the generic level. The following genera were encountered, namely, Vibrio, Bacillus, Alcaligenes, Micrococcus, Pseudomonas, Cytophaga-Flavobacterium, Aeromonas, Corynebacterium and members of Enterobacteriaceae. Vibrio and Bacillus were found to be the dominant groups representing 29.26% and 41.80% respectively of the total isolates. Because of the importance of the Vibrio group in marine environment these isolates were further identified to the species level and it included V. parahaemolyticus, V. alginolyticus, V. consticola, V. anguillarum and V. fischeri. These observations suggest that different groups of arylsulfatase – producing bacteria probably occur in marine sediments.

Disseminação horizontal de genes que codificam para β-lactamases de espectro alargado em isolados de enterobacteriaceae de origem hospitalar

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Estudo da suscetibilidade aos antibióticos em isolados de Enterobacteriaceae provenientes de suiniculturas Portuguesas

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Emergence of CTX-M Group 1-ESBL producing Klebsiella pneumonia from a tertiary care centre in Karachi, Pakistan.

http://www.jidc.org/index.php/journal/article/view/20818098/422 Background: Extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae have been reported previously from Pakistan but the genotypic characteristics of these enzymes is not known. Hence the aim of the study was first to characterise the genotypic content of these beta-lactamases and secondly to assess the clonal relationship of these isolates. Methodology: We analysed 65 non-duplicate ESBL positive, K. pneumoniae isolates prospectively collected based on phenotype as detected using the two-disc method. Isolates were collected from different sources: blood cultures (46.15%; n = 30); tracheal aspirates (24.6%; n = 16); urine (10.7%; n = 7); wound swabs, pus and tissue (18.4%; n = 12). ESBL production was confirmed by the ESBL E-test method and the presence of the blaCTX-M encoding genes was confirmed by polymerase chain reaction. The clonal relationship of clinical isolates was studied by Pulsed Field Gel Electrophoresis. Results: The results showed that 93.84% (n = 61) isolates of K. pneumoniae were positive for the blaCTX-M-1 group. One isolate showed PCR signals for blaCTX-M-25 group. None of our isolates were positive for CTX-M groups 2, 8 and 9. The majority of blaCTX-M positive isolates were genetically unrelated and no epidemic clones were identified. Conclusion: This study reports the emergence of CTX-M groups 1 and 25 producing isolates of K. pneumoniae with genetic diversity in Karachi, Pakistan.

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.

High Prevalence of bla(CTX-M) Extended Spectrum Beta-Lactamase Genes in Klebsiella pneumoniae Isolates from a Tertiary Care Hospital: First report of bla(SHV-12), bla(SHV-31), bla(SHV-38), and bla(CTX-M-15) in Brazil

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

Detecção de beta-lactamase de espectro estendido em membros da família Enterobateriaceae

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Detecção e identificação de genes de β-lactamases de espectro estendido em Salmonella spp. e Escherichia coli isoladas de carnes de frango, suína e bovina destinadas ao consumo humano

Pós-graduação em Microbiologia - IBILCE

Resistência aos beta-lactâmicos e detecção dos genes blashv, blatem, blactx-m e blages em Enterobacteriaceae isoladas de efluentes hospitar e comunitário em um município do noroeste paulista

Pós-graduação em Microbiologia - IBILCE

Avaliação de métodos de triagem e de detecção fenotípica de ß-lactamase de espectro ampliado (ESBL)

The catalytic function of extended-spectrum β-lactamases can result in high degrees of bacterial resistance to β-lactamic antimicrobials and in the emergence of ESBL among the members of Enterobacteriaceae family, especially Klebsiella pneumoniae and Escherichia coli. This occurs due to the dissemination and emergence of new variants of these enzymes caused by the high utilization of antibiotics like broad-spectrum cephalosporins. The ESBL are β-lactamases capable of conferring bacterial resistance to the penicillins, 1st, 2nd and 3rd generation cephalosporins, and aztreonam (but not cephamycins and carbapenems) through the hydrolysis of these antibiotics. In view of this phenomenon, the exact screening and detection of the producers of ESBL are essential for the appropriate selection of the antimicrobial therapy. The purposes of this study were to evaluate the best antimicrobial for the selection of ESBL producers and to determine the best method for the detection of such microorganisms. We evaluated 200 sequential bacterial samples including the species Klebsiella pneumoniae (56.5%), Escherichia coli (34%), Proteus mirabilis (8.5%) and Klebsiella oxytoca (1%), previously characterized as ESBL producers between February and September 2008 in the Laboratory of Microbiology, Botucatu Medical School - UNESP, Botucatu, São Paulo State, Brazil. To select the ESBL-producer bacteria, we used the disks recommended by CLSI 2008, aztreonam (ATM), cefpodoxime (CPD), ceftriaxone (CRO), cefotaxime (CTX) and ceftazidime (CAZ), besides cefepime (FEP). ESBL production was confirmed by three methods: double disk screening, ESBL Etest®, and Vitek® automated system. The disks employed in the double disk screening were: penicillin associated with β-lactamase inhibitor, amoxicillin-clavulanic acid, and two β-lactamic antibiotics, ceftazidime and cefotaxime...(Complete abstract click electronic access below)

Endophytic and rhizospheric enterobacteria isolated from sugar cane have different potentials for producing plant growth-promoting substances

Background and aims Endophytic and rhizospheric environments differ in many respects, leading to the presence of different bacterial communities at each site. However, microorganisms such as enterobacteria can be found both within plants and in the surrounding soil. Bacteria must present differences in the traits that affect such environments in order to successfully colonise them. The present study compared the plant growth-promoting potential of diazotrophic enterobacteria isolated from the rhizosphere and from within surface-disinfected plants. Methods A total of 46 diazotrophic enterobacterial strains (21 rhizospheric and 25 putatively endophytic) belonging to the Klebsiella and Enterobacter genera, which are prevalent in sugar cane plantations, were isolated from the rhizosphere and from surface-disinfected plants. Their ability to synthesise amino acids using combined nitrogen obtained from nitrogen fixation, and their ability to synthesise indole-3-acetic acid (IAA) were determined by high performance liquid chromatography. Endogenous ethylene production by the bacteria was measured using gas chromatography, and biocontrol of phytopathogenic fungi was determined qualitatively using a dual culture technique. Results The putative endophytes released significantly higher amounts of amino acids than the rhizospheric bacteria, whilst the latter produced higher quantities of ethylene and were more actively antagonistic to fungi. Both types of bacteria released similar amounts of IAA. Conclusion Endophytic and rhizospheric bacteria differ in their capacity to release plant growth-promoting substances, which may be a reflection of their adaptations and an indication of their potential impact on their natural environment.

Public health risks of enterobacterial isolates producing extended-spectrum β-lactamases or AmpC β-lactamases in food and food-producing animals: an EU perspective of epidemiology, analytical methods, risk factors, and control options

The blaESBL and blaAmpC genes in Enterobacteriaceae are spread by plasmid-mediated integrons, insertion sequences, and transposons, some of which are homologous in bacteria from food animals, foods, and humans. These genes have been frequently identified in Escherichia coli and Salmonella from food animals, the most common being blaCTX-M-1, blaCTX-M-14, and blaCMY-2. Identification of risk factors for their occurrence in food animals is complex. In addition to generic antimicrobial use, cephalosporin usage is an important risk factor for selection and spread of these genes. Extensive international trade of animals is a further risk factor. There are no data on the effectiveness of individual control options in reducing public health risks. A highly effective option would be to stop or restrict cephalosporin usage in food animals. Decreasing total antimicrobial use is also of high priority. Implementation of measures to limit strain dissemination (increasing farm biosecurity, controls in animal trade, and other general postharvest controls) are also important.

Emergence of Klebsiella pneumoniae co-producing NDM-1, OXA-48, CTX-M-15, CMY-16, QnrA and ArmA in Switzerland

Extensively drug-resistant (XDR) Klebsiella pneumoniae isolates usually carry a single carbapenemase (e.g. KPC, NDM, OXA-48-like). Here we describe an XDR K. pneumoniae of sequence type 101 that was detected in the screening rectal swab of a patient transferred from the intensive care unit of a hospital located in Belgrade (Serbia) to Bern University Hospital (Switzerland). The isolate was resistant to all antibiotics with the exception of colistin [minimum inhibitory concentration] (MIC≤0.125μg/mL), tigecycline (MIC=0.5μg/mL) and fosfomycin (MIC=2μg/mL). The isolate co-possessed class B (NDM-1) and class D (OXA-48) carbapenemases, class A extended-spectrum β-lactamase (CTX-M-15), class C cephalosporinase (CMY-16), ArmA 16S rRNA methyltransferase, substitutions in GyrA and ParC, loss of OmpK35 porin, as well as other genes conferring resistance to quinolones (qnrA), tetracyclines [tet(A)], sulfonamides (sul1, sul2), trimethoprim (dfrA12, dfrA14), rifampicin (arr-1), chloramphenicol (cmlA1, floR) and streptomycin (aadA1). The patient was placed under contact isolation precautions preventing the spread of this nearly untreatable pathogen.