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1.
Appl Environ Microbiol ; 88(13): e0046522, 2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35736227

RESUMO

Wildlife play a role in the acquisition, maintenance, and dissemination of antimicrobial resistance (AMR). This is especially true at the human-domestic animal-wildlife interface, like urbanized areas, where interactions occur that can promote the cross-over of AMR bacteria and genes. We conducted a 2-year fecal surveillance (n = 783) of a white-tailed deer (WTD) herd from an urban park system in Ohio to identify and characterize cephalosporin-resistant and carbapenemase-producing bacteria using selective enrichment. Using generalized linear mixed models we found that older (OR = 2.3, P < 0.001), male (OR = 1.8, P = 0.001) deer from urbanized habitats (OR = 1.4, P = 0.001) were more likely to harbor extended-spectrum cephalosporin-resistant Enterobacterales. In addition, we isolated two carbapenemase-producing Enterobacterales (CPE), a Klebsiella quasipneumoniae harboring blaKPC-2 and an Escherichia coli harboring blaNDM-5, from two deer from urban habitats. The genetic landscape of the plasmid carrying blaKPC-2 was unique, not clustering with other reported plasmids encoding KPC-2, and only sharing 78% of its sequence with its nearest match. While the plasmid carrying blaNDM-5 shared sequence similarity with other reported plasmids encoding NDM-5, the intact IS26 mobile genetic elements surrounding multiple drug resistant regions, including the blaNDM-5, has been reported infrequently. Both carbapenemase genes were successfully conjugated to a J53 recipient conferring a carbapenem-resistant phenotype. Our findings highlight that urban environments play a significant role on the transmission of AMR bacteria and genes to wildlife and suggest WTD may play a role in the dissemination of clinically and epidemiologically relevant antimicrobial resistant bacteria. IMPORTANCE The role of wildlife in the spread of antimicrobial resistance is not fully characterized. Some wildlife, including white-tailed deer (WTD), can thrive in suburban and urban environments. This may result in the exchange of antimicrobial resistant bacteria and resistance genes between humans and wildlife, and lead to their spread in the environment. We found that WTD living in an urban park system carried antimicrobial resistant bacteria that were important to human health and resistant to antibiotics used to treat serious bacterial infections. This included two deer that carried bacteria resistant to carbapenem antibiotics which are critically important for treatment of life-threatening infections. These two bacteria had the ability to transfer their AMR resistance genes to other bacteria, making them a threat to public health. Our results suggest that WTD may contribute to the spread of antimicrobial resistant bacteria in the environment.


Assuntos
Cefalosporinase , Cervos , Farmacorresistência Bacteriana , Gammaproteobacteria/isolamento & purificação , Animais , Animais Selvagens/microbiologia , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Cefalosporinase/genética , Cefalosporinas/farmacologia , Cervos/microbiologia , Gammaproteobacteria/efeitos dos fármacos , Humanos , Masculino , Testes de Sensibilidade Microbiana , Plasmídeos
2.
Anal Chem ; 94(26): 9442-9449, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35748103

RESUMO

Infections caused by bacteria that produce ß-lactamases (BLs) are a major problem in hospital settings. The phenotypic detection of these bacterial strains requires culturing samples prior to analysis. This procedure may take up to 72 h, and therefore it cannot be used to guide the administration of the first antibiotic regimen. Here, we propose a multisensor for identifying pathogens bearing different types of ß-lactamases above the infectious dose threshold within 90 min that does not require culturing samples. Instead, bacterial cells are preconcentrated in the cellulose scaffold of a paper-based multisensor. Then, 12 assays are performed in parallel to identify whether the pathogens produce carbapenemases and/or cephalosporinases, including metallo-ß-lactamases, extended-spectrum ß-lactamases (ESBLs), and AmpC enzymes. The multisensor generates an array of colored spots that can be quantified with image processing software and whose interpretation leads to the detection of the different enzymes depending on their specificity toward the hydrolysis of certain antibiotics, and/or their pattern of inhibition or cofactor activation. The test was validated for the diagnosis of urinary tract infections. The inexpensive paper platform along with the uncomplicated colorimetric readout makes the proposed prototypes promising for developing fully automated platforms for streamlined clinical diagnosis.


Assuntos
Cefalosporinase , Colorimetria , Antibacterianos/farmacologia , Bactérias , Proteínas de Bactérias , Testes de Sensibilidade Microbiana , beta-Lactamases/análise
3.
Int J Antimicrob Agents ; 59(6): 106595, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35483625

RESUMO

BACKGROUND: Treatment options for metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa infections are limited. Imipenem/relebactam (I/R) plus aztreonam (ATM) may be an option. METHODS: Ten OprD(-) P. aeruginosa isolates (3 parent strains; 7 MBL-producers) were evaluated using checkerboard methodology and Fractional Inhibitory Concentration Index (FICI). Isolates exhibiting synergy in checkerboard studies (FICI ≤0.5) were evaluated using 24-h static concentration time-kill. Bacteria in late log-phase growth were diluted to 1 × 106 cfu/mL and incubated at 37°C for 24 h. Samples were drawn at 0, 2, 4, 6 and 24 h. Physiological fCmax, fCss,avg and fCmin of imipenem (26.7, 5.6, 0.5 mg/L), relebactam (REL; 13.1, 4, 0.8 mg/L) and ATM (62, 29, 8 mg/L) were used. Synergy in time-kill studies was defined as >2 log10 cfu/mL reduction compared with the most active individual agent. RESULTS: Synergy was observed in five isolates in checkerboard studies, including three of seven MBL-producing isolates. Isolates that were OprD(-) and harbored inducible Pseudomonas-derived cephalosporinases (PDCs) did not show synergy as defined by FICI; however, ATM minimum inhibitory concentrations (MICs) were significantly reduced with the combination. In time-kill studies, ATM alone was as active as combination regimens for MBL-producing isolates with deleted or inducible PDC production. For strains exhibiting constitutive PDC production, I/R plus ATM was synergistic at fCss,avg concentrations but exhibited similar activity to ATM at fCmin and fCmax concentrations. CONCLUSIONS: I/R plus ATM appears to exhibit synergy for some MBL-producing P. aeruginosa at physiological concentrations. Further study of the effect of dynamic concentrations is needed to fully understand the utility of this combination.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Compostos Azabicíclicos , Aztreonam/farmacologia , Cefalosporinase , Humanos , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Pseudomonas , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , beta-Lactamases/farmacologia
4.
Microbiol Spectr ; 10(1): e0201921, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35171032

RESUMO

In the current scenario of antibiotic resistance magnification, new weapons against top nosocomial pathogens like Pseudomonas aeruginosa are urgently needed. The interplay between ß-lactam resistance and virulence is considered a promising source of targets to be attacked by antivirulence therapies, and in this regard, we previously showed that a peptidoglycan recycling blockade dramatically attenuated the pathogenic power of P. aeruginosa strains hyperproducing the chromosomal ß-lactamase AmpC. Here, we sought to ascertain whether this observation could be applicable to other ß-lactamases. To do so, P. aeruginosa wild-type or peptidoglycan recycling-defective strains (ΔampG and ΔnagZ) harboring different cloned ß-lactamases (transferable GES, VIM, and OXA types) were used to assess their virulence in Galleria mellonella larvae by determining 50% lethal doses (LD50s). A mild yet significant LD50 increase was observed after peptidoglycan recycling disruption per se, whereas the expression of class A and B enzymes did not impact virulence. While the production of the narrow-spectrum class D OXA-2 entailed a slight attenuation, its extended-spectrum derivatives OXA-226 (W159R [bearing a change of W to R at position 159]), OXA-161 (N148D), and principally, OXA-539 (D149 duplication) were associated with outstanding virulence impairments, especially in recycling-defective backgrounds (with some LD50s being >1,000-fold that of the wild type). Although their exact molecular bases remain to be deciphered, these results suggest that mutations affecting the catalytic center and, therefore, the hydrolytic spectrum of OXA-2-derived enzymes also drastically impact the pathogenic power of P. aeruginosa. This work provides new and relevant knowledge to the complex topic of the interplay between the production of ß-lactamases and virulence that could be useful to build future therapeutic strategies against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is one of the leading nosocomial pathogens whose growing resistance makes the development of therapeutic options extremely urgent. The resistance-virulence interplay has classically aroused researchers' interest as a source of therapeutic targets. In this regard, we describe a wide array of virulence attenuations associated with different transferable ß-lactamases, among which the production of OXA-2-derived extended-spectrum ß-lactamases stood out as a dramatic handicap for pathogenesis, likely as a side effect of mutations causing the expansion of their hydrolytic spectrums. Moreover, our results confirm the validity of disturbing peptidoglycan recycling as a weapon to attenuate P. aeruginosa virulence in class C and D ß-lactamase production backgrounds. In the current scenario of dissemination of horizontally acquired ß-lactamases, this work brings out new data on the complex interplay between the production of specific enzymes and virulence attenuation that, if complemented with the characterization of the underlying mechanisms, will likely be exploitable to develop future virulence-targeting antipseudomonal strategies.


Assuntos
Peptidoglicano/efeitos dos fármacos , Peptidoglicano/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Cefalosporinase , Transferência Genética Horizontal , Proteínas de Membrana Transportadoras , Testes de Sensibilidade Microbiana , Mariposas , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Virulência/efeitos dos fármacos , Resistência beta-Lactâmica/efeitos dos fármacos , Resistência beta-Lactâmica/genética
5.
Antimicrob Agents Chemother ; 65(8): e0008921, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34060900

RESUMO

A ceftolozane-tazobactam- and ceftazime-avibactam-resistant Pseudomonas aeruginosa isolate was recovered after treatment (including azithromycin, meropenem, and ceftolozane-tazobactam) from a patient that had developed ventilator-associated pneumonia after COVID-19 infection. Whole-genome sequencing revealed that the strain, belonging to ST274, had acquired a nonsense mutation leading to truncated carbapenem porin OprD (W277X), a 7-bp deletion (nt213Δ7) in NfxB (negative regulator of the efflux pump MexCD-OprJ), and two missense mutations (Q178R and S133G) located within the first large periplasmic loop of MexD. Through the construction of mexD mutants and complementation assays with wild-type nfxB, it was evidenced that resistance to the novel cephalosporin-ß-lactamase inhibitor combinations was caused by the modification of MexD substrate specificity.


Assuntos
COVID-19 , Infecções por Pseudomonas , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cefalosporinase , Cefalosporinas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Pseudomonas , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/genética , SARS-CoV-2 , Inibidores de beta-Lactamases/farmacologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-33782013

RESUMO

Ceftazidime (CAZ)-avibactam (AVI) is a ß-lactam/ß-lactamase inhibitor combination with activity against type A and type C ß-lactamases. Resistance emergence has been seen, with multiple mechanisms accounting for the resistance. We performed four experiments in the dynamic hollow-fiber infection model, delineating the linkage between drug exposure and both the rate of bacterial kill and resistance emergence by all mechanisms. The Pseudomonas aeruginosa isolate had MICs of 1.0 mg/liter (CAZ) and 4 mg/liter (AVI). We demonstrated that the time at ≥4.0 mg/liter AVI was linked to the rate of bacterial kill. Linkage to resistance emergence/suppression was more complex. In one experiment in which CAZ and AVI administration was intermittent and continuous, respectively, and in which AVI was given in unitary steps from 1 to 8 mg/liter, AVI at up to 3 mg/liter allowed resistance emergence, whereas higher values did not. The threshold value was 3.72 mg/liter as a continuous infusion to counterselect resistance (AVI area under the concentration-time curve [AUC] of 89.3 mg · h/liter). The mechanism involved a 7-amino-acid deletion in the Ω-loop region of the Pseudomonas-derived cephalosporinase (PDC) ß-lactamase. Further experiments in which CAZ and AVI were both administered intermittently with regimens above and below the AUC of 89.3 mg · h/liter resulted in resistance in the lower-exposure groups. Deletion mutants were not identified. Finally, in an experiment in which paired exposures as both continuous and intermittent infusions were performed, the lower value of 25 mg · h/liter by both profiles allowed selection of deletion mutants. Of the five instances in which these mutants were recovered, four had a continuous-infusion profile. Both continuous-infusion administration and low AVI AUC exposures have a role in selection of this mutation.


Assuntos
Ceftazidima , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Compostos Azabicíclicos/farmacologia , Ceftazidima/farmacologia , Cefalosporinase , Combinação de Medicamentos , Testes de Sensibilidade Microbiana , Pseudomonas , Pseudomonas aeruginosa/genética
7.
Artigo em Inglês | MEDLINE | ID: mdl-33486218

RESUMO

The screening and identification of bioactive components, which are effectively resistant to metallo-beta-lactamase (MßL), were studied in the alcohol extract of Schisandra chinensis (Turcz.) Baill. by metalloenzyme-immobilized affinity chromatography. Taking bizinc metalloenzyme beta-lactamase II from Bacillus cereus (Bc II) and monozinc metalloenzyme CphA from aeromonas hydrophila (CphA) as examples, we studied the feasibility of this scheme based on the construction of metalloenzyme-immobilized chromatographic model. It was found that the Bc II- and CphA-immobilized chromatographic column could be used not only to explore the interaction between the MßL and their specific ligands, but also to screen the bioactive components from traditional Chinese medicine. The Bc II-and CphA-immobilized columns were used to screen the bioactive components from the alcohol extract of Schisandra chinensis (Turcz.) Baill. Time-of-flight tandem mass spectrometry analysis and molecular docking revealed that isobutyl 3-O-sulfo-ß-D-galactopyranoside is the effective bioactive components that could bind with metalloenzyme Bc II. It is believed that our current work may provide a methodological reference for screening MßL inhibitors from traditional Chinese medicine.


Assuntos
Proteínas de Bactérias/metabolismo , Cefalosporinase/metabolismo , Descoberta de Drogas/métodos , Extratos Vegetais/análise , Schisandra/química , beta-Lactamases/metabolismo , Proteínas de Bactérias/química , Cefalosporinase/química , Cromatografia de Afinidade , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Galactosídeos/análise , Galactosídeos/química , Galactosídeos/metabolismo , Simulação de Acoplamento Molecular , Extratos Vegetais/química , Extratos Vegetais/metabolismo , beta-Lactamases/química
8.
Int J Antimicrob Agents ; 57(2): 106271, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33352235

RESUMO

AIMS: Polymyxin-based combination therapy is often used to treat carbapenem-resistant Acinetobacter baumannii (A. baumannii) infections. Although sulbactam is intrinsically active against A. baumannii, few studies have investigated colistin/sulbactam combinations against carbapenem-resistant A. baumannii. METHODS: Whole genome sequencing was undertaken on eight carbapenem-resistant (colistin-susceptible) isolates of A. baumannii from Chinese patients. Bacterial killing of colistin and sulbactam, alone and in combination, was examined with checkerboard (all isolates) and static and dynamic time-kill studies (three isolates). In the dynamic studies, antibiotics were administered in various clinically-relevant dosing regimens that mimicked patient pharmacokinetics. RESULTS: The eight isolates consisted of ST195, ST191 and ST208 belonging to clonal complex 208, which is the most epidemic clonal type of A. baumannii globally. All isolates possessed Acinetobacter-derived cephalosporinase (ADC-61 or ADC-78) and seven of eight isolates contained the carbapenem-resistance gene blaOXA-23. The colistin/sulbactam combination was synergistic against two of eight isolates in checkerboard studies. In time-kill studies, rapid bacterial killing of ca. 3-6 log10 CFU/mL was observed with colistin monotherapy, followed by steady regrowth. Sulbactam monotherapy was generally ineffective. Substantially enhanced bacterial killing was observed with colistin/sulbactam combinations in both static and dynamic models, especially with the higher sulbactam concentration (2 g) and/or longer sulbactam infusion time (2 hours) in the dynamic model. CONCLUSIONS: This study was the first to use a pharmacokinetics/pharmacodynamics model to investigate synergistic activity of colistin/sulbactam combinations against A. baumannii. It showed that clinically-relevant dosing regimens of colistin combined with sulbactam may substantially improve bacterial killing of multidrug-resistant and carbapenem-resistant A. baumannii.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Colistina/farmacologia , Sulbactam/farmacologia , Resistência beta-Lactâmica , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacocinética , Cefalosporinase/genética , Colistina/farmacocinética , Combinação de Medicamentos , Sinergismo Farmacológico , Genoma Bacteriano , Humanos , Testes de Sensibilidade Microbiana , Sulbactam/farmacocinética , Sequenciamento Completo do Genoma , Resistência beta-Lactâmica/genética
10.
Antimicrob Agents Chemother ; 64(12)2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-32988830

RESUMO

Extended-spectrum class C ß-lactamases have evolved to rapidly inactivate expanded-spectrum cephalosporins, a class of antibiotics designed to be resistant to hydrolysis by ß-lactamase enzymes. To better understand the mechanism by which Acinetobacter-derived cephalosporinase-7 (ADC-7), a chromosomal AmpC enzyme, hydrolyzes these molecules, we determined the X-ray crystal structure of ADC-7 in an acyl-enzyme complex with the cephalosporin ceftazidime (2.40 Å) as well as in complex with a boronic acid transition state analog inhibitor that contains the R1 side chain of ceftazidime (1.67 Å). In the acyl-enzyme complex, the carbonyl oxygen is situated in the oxyanion hole where it makes key stabilizing interactions with the main chain nitrogens of Ser64 and Ser315. The boronic acid O1 hydroxyl group is similarly positioned in this area. Conserved residues Gln120 and Asn152 form hydrogen bonds with the amide group of the R1 side chain in both complexes. These complexes represent two steps in the hydrolysis of expanded-spectrum cephalosporins by ADC-7 and offer insight into the inhibition of ADC-7 by ceftazidime through displacement of the deacylating water molecule as well as blocking its trajectory to the acyl carbonyl carbon. In addition, the transition state analog inhibitor, LP06, was shown to bind with high affinity to ADC-7 (Ki , 50 nM) and was able to restore ceftazidime susceptibility, offering the potential for optimization efforts of this type of inhibitor.


Assuntos
Acinetobacter , Ácidos Borônicos , Ceftazidima , Cefalosporinase , Antibacterianos/farmacologia , Ácidos Borônicos/farmacologia , Ceftazidima/farmacologia , Cefalosporinase/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases
11.
Sci Rep ; 10(1): 10167, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576927

RESUMO

Yersinia enterocolitica is an enteric bacterium which can cause severe gastroenteritis. Beta-lactams are the most widely used antibiotics against Y. enterocolitica. Y. enterocolitica produces two chromosomal ß-lactamases, BlaA and BlaB. BlaB is an Ambler Class C inducible broad spectrum cephlaosporinase which showed differential enzyme activity in different biotypes of Y. enterocolitica. The expression of blaB is mainly regulated by ampR- the transcriptional regulator and, ampD - which helps in peptidoglycan recycling. The aim of this study was to identify and characterize genetic determinants underlying differential enzyme activity of BlaB in Y. enterocolitica biotypes 1 A, IB, 2 and 4. Thus, ampR, blaB and ampD were PCR-amplified and modeled in silico. The intercistronic region containing promoters of ampR and blaB was also investigated. Our results indicated that blaB was more inducible in biotypes 2 and 4, than in biotypes 1 A and 1B. Superimposition of in silico modeled proteins suggested that variations in amino acid sequences of AmpR, BlaB and AmpD were not responsible for hyper-production of BlaB in biotypes 2 and 4. Analysis of promoter regions of ampR and blaB revealed variations at -30, -37 and -58 positions from blaB transcription start site. Studies on relative expression levels of blaB in different biotypes by qRT-PCR indicated that nucleotide variations at these positions might contribute to a higher enzyme activity of BlaB in biotypes 2 and 4. However, this is a preliminary study and further studies including more strains of each biotype are required to strengthen our findings. Nevertheless, to the best of our knowledge, this is the first study which has investigated the genetic determinants underlying differential inducible production of BlaB in different biotypes of Y. enterocolitica.


Assuntos
Cefalosporinase/genética , Cefalosporinase/metabolismo , Regulação Bacteriana da Expressão Gênica , Expressão Gênica , Yersinia enterocolitica/citologia , Yersinia enterocolitica/enzimologia , Proteínas de Bactérias/fisiologia , Peptidoglicano/metabolismo , Yersinia enterocolitica/metabolismo , Yersinia enterocolitica/patogenicidade
12.
ACS Infect Dis ; 6(7): 1965-1975, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32502340

RESUMO

Boronic acid transition state inhibitors (BATSIs) are known reversible covalent inhibitors of serine ß-lactamases. The selectivity and high potency of specific BATSIs bearing an amide side chain mimicking the ß-lactam's amide side chain are an established and recognized synthetic strategy. Herein, we describe a new class of BATSIs where the amide group is replaced by a bioisostere triazole; these compounds were designed as molecular probes. To this end, a library of 26 α-triazolylmethaneboronic acids was synthesized and tested against the clinically concerning Acinetobacter-derived cephalosporinase, ADC-7. In steady state analyses, these compounds demonstrated Ki values ranging from 90 nM to 38 µM (±10%). Five compounds were crystallized in complex with ADC-7 ß-lactamase, and all the crystal structures reveal the triazole is in the putative amide binding site, thus confirming the triazole-amide bioisosterism. The easy synthetic access of these new inhibitors as prototype scaffolds allows the insertion of a wide range of chemical groups able to explore the enzyme binding site and provides insights on the importance of specific residues in recognition and catalysis. The best inhibitor identified, compound 6q (Ki 90 nM), places a tolyl group near Arg340, making favorable cation-π interactions. Notably, the structure of 6q does not resemble the natural substrate of the ß-lactamase yet displays a pronounced inhibition activity, in addition to lowering the minimum inhibitory concentration (MIC) of ceftazidime against three bacterial strains expressing class C ß-lactamases. In summary, these observations validate the α-triazolylboronic acids as a promising template for further inhibitor design.


Assuntos
Acinetobacter baumannii , Inibidores de beta-Lactamases , Acinetobacter baumannii/metabolismo , Cefalosporinase/genética , Cefalosporinase/metabolismo , Relação Estrutura-Atividade , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/metabolismo
13.
Acta Vet Scand ; 62(1): 18, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-32334616

RESUMO

Sweden has a long tradition of monitoring occurrence of antibiotic resistant bacteria in both animals and humans, but there currently is no organised and harmonized monitoring on carriage of Enterobacteriaceae producing extended-spectrum beta-lactamase (ESBL), plasmid-mediated AmpC beta-lactamase (pAmpC), or methicillin-resistant coagulase positive staphylococci e.g. methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs. The aim of the current study was therefore to determine the prevalence of ESBL/pAmpC producing Enterobacteriaceae and methicillin-resistant coagulase positive staphylococci in healthy dogs in Sweden, and to phenotypically and genotypically characterize any identified isolates. It was shown that 0.9% (95% confident interval 0.3-2.7%) of the dogs (n = 325) carried multi-resistant ESBL-producing Escherichia coli, but that no methicillin-resistant coagulase positive staphylococci could be detected. In conclusion, the occurrence of multi-drug resistant bacteria remains rare among healthy dogs in Sweden. In addition, the ESBL-producing E. coli identified showed genetic characteristics related to those reported from humans.


Assuntos
Doenças do Cão/microbiologia , Cães/microbiologia , Farmacorresistência Bacteriana Múltipla , Infecções por Enterobacteriaceae/veterinária , Enterobacteriaceae/isolamento & purificação , Animais , Antibacterianos/farmacologia , Cefalosporinase/metabolismo , Doenças do Cão/epidemiologia , Enterobacteriaceae/classificação , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/microbiologia , Genótipo , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Suécia/epidemiologia
14.
Res Microbiol ; 171(3-4): 128-133, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31988011

RESUMO

A wide range of intrinsic Acinetobacter-derived cephalosporinases (ADC) along with other carbapenemases has now been detected in Acinetobacter baumannii leaving clinicians with few treatment options. The present study reports the spread of ADC-30 co-producing KPC-2 along with other ß-lactamases among carbapenem resistant A. baumannii strains obtained from ICU patients in two Indian hospitals. Primer extension analysis revealed higher transcript level of the ADC gene when induced with cefoxitin at 8 µg/ml (170 fold), ceftriaxone at 8 µg/ml (136 fold), ceftazidime at 4 µg/ml (65 fold), cefepime at 8 µg/ml (77 fold) and aztreonam at 8 µg/ml (21 fold) when compared with the basal level without antibiotic pressure. Slight increase in expression of blaADC-30 when induced with imipenem and meropenem at 0.25 µg/ml (3 and 6 fold) was observed and may help in conferring resistance to carbapenem. MLST analysis revealed the circulation of A. baumannii sequence types ST188, ST386, ST583 and ST390 in these hospitals.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Cefalosporinase/genética , Resistência beta-Lactâmica , Acinetobacter baumannii/classificação , Farmacorresistência Bacteriana Múltipla , Regulação Bacteriana da Expressão Gênica , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Análise de Sequência de DNA
16.
Anal Chem ; 91(17): 11316-11323, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31403771

RESUMO

The traditional enzyme-linked immunosorbent assay (ELISA) has some disadvantages, such as insufficient sensitivity and low stability of the labeled enzyme, which limit its further applications. In this study, a more stable enzyme, Amp cephalosporinase (AmpC), was selected as the labeled enzyme, and its substrate was designed and synthesized. This substrate contained the cephalosporin ring core as the enzymatic recognition section and the structural motif of the 3-hydroxyflavone (3-HF) as the reporter molecule. AmpC can specifically catalyze the substrate and release 3-HF, which can enter the cavity of ß-cyclodextrin (ß-CD) on the surface of ZnS quantum dots and form a fluorescence resonance energy transfer (FRET) signal amplification system. An AmpC-catalyzed, FRET-mediated ultrasensitive immunosensor (ACF immunosensor) for procalcitonin (PCT) was developed by combining the signal amplification system of the polystyrene microspheres and effective immune-based magnetic separation. The ACF immunosensor has high sensitivity and specificity for the detection of PCT: its linear range is from 0.1 ng mL-1 to 70 ng mL-1, and the limit of detection can reach 0.03 ng mL-1. The spiking recoveries of PCT in human serum samples range from 98.3% to 107%, with relative standard deviations ranging from 2.14% to 12.0%. This approach was applied to detect PCT in real patient serum samples, and the results are consistent with those obtained with a commercial ELISA kit.


Assuntos
Técnicas Biossensoriais , Cefalosporinase/química , Ensaio de Imunoadsorção Enzimática , Flavonoides/sangue , Transferência Ressonante de Energia de Fluorescência , Cefalosporinase/síntese química , Cefalosporinase/metabolismo , Humanos , Estrutura Molecular , Especificidade por Substrato
17.
Zoonoses Public Health ; 66(6): 603-617, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31264805

RESUMO

OBJECTIVES: This study investigates the frequency and characteristics of carbapenemase-producing Escherichia coli/Klebsiella pneumoniae (CPE/K) and extended-spectrum cephalosporinase-producing E. coli/K. pneumoniae (ESCE/K) in healthy humans and livestock in rural Cambodia. Additionally, household practices as risk factors for faecal carriage of ESCE/K are identified. METHODS: Faecal samples were obtained from 307 humans and 285 livestock including large ruminants, pigs and poultry living in 100 households in rural Cambodia in 2011. Each household was interviewed, and multilevel logistic model determined associations between household practices/meat consumption and faecal carriage of ESCE/K. CPE and ESCE/K were detected and further screened for colistin resistance genes. RESULTS: CPE/K isolates harbouring blaOXA-48 were identified in two humans. The community carriage of ESCE/K was 20% in humans and 23% in livestock. The same ESBL genes: blaCTX-M-15 , blaCTX-M-14 , blaCTX-M-27 , blaCTX-M-55 , blaSHV-2 , blaSHV-12 , blaSHV-28 ; AmpC genes: blaCMY-2 , blaCMY-42, blaDHA-1 ; and colistin resistance genes: mcr-1-like and mcr-3-like were detected in humans and livestock. ESCE/K was frequently detected in women, young children, pigs and poultry, which are groups in close contact. The practice of burning or burying meat waste and not collecting animal manure indoors and outdoors daily were identified as risk factors for faecal carriage of ESCE/K. CONCLUSIONS: Faecal carriage of E. coli and K. pneumoniae harbouring extended-spectrum cephalosporinase genes are common in the Cambodian community, especially in women and young children. Exposure to animal manure and slaughter products are risk factors for intestinal colonization of ESCE/K in humans.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Cefalosporinase/metabolismo , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamases/metabolismo , Adolescente , Adulto , Animais , Proteínas de Bactérias/genética , Camboja , Cefalosporinase/genética , Criança , Pré-Escolar , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla , Escherichia coli/enzimologia , Fezes/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Lactente , Klebsiella pneumoniae/enzimologia , Gado/microbiologia , Masculino , Fatores de Risco , População Rural , Zoonoses , beta-Lactamases/genética
18.
PLoS One ; 14(5): e0217174, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31100096

RESUMO

Extended-spectrum ß-lactamase (ESBL)- and plasmid mediated AmpC-type cephalosporinase (pAmpC)-producing Escherichia coli (ESBL/pAmpC E. coli) in food-producing animals is a major public health concern. This study aimed at quantifying ESBL/pAmpC-E. coli occurrence and transfer in Italy's broiler production pyramid. Three production chains of an integrated broiler company were investigated. Cloacal swabs were taken from parent stock chickens and offspring broiler flocks in four fattening farms per chain. Carcasses from sampled broiler flocks were collected at slaughterhouse. Samples were processed on selective media, and E. coli colonies were screened for ESBL/pAmpC production. ESBL/pAmpC genes and E. coli phylogroups were determined by PCR and sequencing. Average pairwise overlap of ESBL/pAmpC E. coli gene and phylogroup occurrences between subsequent production stages was estimated using the proportional similarity index, modelling uncertainty in a Monte Carlo simulation setting. In total, 820 samples were processed, from which 513 ESBL/pAmpC E. coli isolates were obtained. We found a high prevalence (92.5%, 95%CI 72.1-98.3%) in day-old parent stock chicks, in which blaCMY-2 predominated; prevalence then dropped to 20% (12.9-29.6%) at laying phase. In fattening broilers, prevalence was 69.2% (53.6-81.3%) at the start of production, 54.2% (38.9-68.6%) at slaughter time, and 61.3% (48.1-72.9%) in carcasses. Significantly decreasing and increasing trends for respectively blaCMY-2 and blaCTX-M-1 gene occurrences were found across subsequent production stages. ESBL/pAmpC E. coli genetic background appeared complex and bla-gene/phylogroup associations indicated clonal and horizontal transmission. Modelling revealed that the average transfer of ESBL/pAmpC E. coli genes between subsequent production stages was 47.7% (42.3-53.4%). We concluded that ESBL/pAmpC E. coli in the broiler production pyramid is prevalent, with substantial transfer between subsequent production levels.


Assuntos
Proteínas de Bactérias/metabolismo , Cefalosporinase/metabolismo , Galinhas/microbiologia , Infecções por Escherichia coli/veterinária , Transmissão Vertical de Doenças Infecciosas/veterinária , Doenças das Aves Domésticas/epidemiologia , Doenças das Aves Domésticas/transmissão , beta-Lactamases/metabolismo , Animais , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/transmissão , Plasmídeos , Doenças das Aves Domésticas/microbiologia
19.
Gene ; 704: 25-30, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980942

RESUMO

Yersinia enterocolitica strains produce two chromosomal ß­lactamases, BlaA - a constitutively produced penicillinase, and BlaB - an inducible "AmpC-type" cephalosporinase. As in other members of Enterobacteriaceae, expression of ampC in Y. enterocolitica is regulated by the genes - ampR and ampD. The ampR encodes a transcriptional regulator which represses the expression of ampC and, ampD encodes a cytoplasmic N­acetyl­anhydromuramyl­l­alanine amidase which participates in recycling of peptidoglycan. Exposure of bacteria to antibiotics like imipenem and cefoxitin results in generation and accumulation of large quantities of muropeptides in cytoplasm which is beyond the recycling capability of AmpD. These muropeptides bind to AmpR, converting it into an activator of ampC expression (ampC de-repression). Earlier studies from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A strains produced a "BlaB-like" enzyme which was non-heterogeneous and showed a differential expression when induced with imipenem. The detection of "BlaB-like" cephalosporinase which was also induced differentially in Y. enterocolitica biotype 1A strains presented an opportunity to discern newer mechanisms, if any, which may underlie inducible expression of "AmpC-type" cephalosporinases. Thus, the objective of the present study was to understand the role of ampR and ampD in regulating differential expression of "BlaB-like" cephalosporinases in biotype 1A strains. Analysis of promoters and amino acid sequences of AmpR revealed that these were conserved in all strains of biotype 1A. Analysis of AmpD amino acid sequences revealed that five variants of AmpD were present which did not contribute to hyper-inducible production of "BlaB-like" enzyme. In-silico prediction of the mRNA secondary structures of ampD revealed significant differences, which might have affected the rate of translation of ampD and accumulation of un-recycled muropeptides inside the cell leading to hyper production of "BlaB-like" cephalosporinases in some Y. enterocolitica biotype 1A strains. The findings provide newer insights to our understanding of the mechanisms underlying regulation of expression of "AmpC-type" ß­lactamases.


Assuntos
Proteínas de Bactérias/genética , Cefalosporinase/genética , N-Acetil-Muramil-L-Alanina Amidase/genética , Yersinia enterocolitica/genética , Clonagem Molecular , Indução Enzimática/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Homologia de Sequência , Yersinia enterocolitica/classificação , Yersinia enterocolitica/enzimologia , beta-Lactamases/genética
20.
Microb Drug Resist ; 25(6): 824-829, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30817214

RESUMO

Yersinia enterocolitica biotype 1A strains are emerging pathogens, frequently isolated from clinical samples across the globe. Y. enterocolitica strains produce two chromosomal ß-lactamases, BlaA and BlaB. BlaA is a constitutively expressed, Ambler class A, penicillinase, whereas BlaB is Ambler class C-type, inducible cephalosporinase. An earlier study from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A produced a "BlaB-like" enzyme. The objective of this work was to study the molecular characteristics of "Bla-B like" ß-lactamases produced by biotype 1A strains to discern their similarity with AmpC-type ß-lactamases and the basis of varied levels of minimum inhibitory concentration (MIC) to cefotaxime. Thus, the promoters and blaB genes were investigated in four strains of biotype 1A. Three-dimensional structures of the "BlaB-like" enzymes were modeled, and docked in silico with cefotaxime to understand how specific substitutions in gene sequences affect antibiotic susceptibilities. Our results indicated that all the reported key catalytic residues were present in variants of "Bla-B-like" enzymes, discerned in biotype 1A strains, but at different positions. Molecular docking of enzyme variants with cefotaxime revealed that lesser was the number of the H-binding residues with cefotaxime in a strain, lower was the MIC of cefotaxime in that strain. To the best of our knowledge, this is the first study in which the molecular characteristics and enzymatic interactions of "BlaB-like" cephalosporinases of Y. enterocolitica biotype 1A strains have been reported.


Assuntos
Cefalosporinase/genética , Cromossomos Bacterianos/genética , Yersinia enterocolitica/genética , Cefotaxima/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Testes de Sensibilidade Microbiana/métodos , Simulação de Acoplamento Molecular , Penicilinase/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Yersinia enterocolitica/efeitos dos fármacos , beta-Lactamases/genética
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