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1.
BMC Microbiol ; 24(1): 168, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38760674

RESUMO

BACKGROUND: We aimed to compare the performance of carbapenemase classification in carbapenem-resistant Klebsiella pneumoniae (CRKP) obtained using the BD Phoenix CPO Detect panel (CPO panel) and Cepheid Xpert Carba-R assays. We analyzed 55 CRKP strains from clinical specimens collected between November 2020 and November 2022. The CPO panel was used to detect both antibiotic susceptibility and phenotypic carbapenemase classes, while Xpert Carba-R was employed to identify KPC, NDM, VIM, OXA-48, and IMP genes. Due to the limited availability of molecular kits, we arbitrarily selected 55 isolates, identified as carbapenemase-producing according to the CPO panel and with meropenem minimum inhibitory concentration values > 8 mg/L. RESULTS: According to the Xpert Carba-R assay, 16 of the 55 isolates (29.1%) were categorised as Ambler Class A (11 of which matched CPO panel Class A identification); three isolates (5.5%) were identified as Class B and 27 isolates (49.1%) as Class D (in both cases consistent with CPO panel B and D classifications). A further eight isolates (14.5%) exhibited multiple carbapenemase enzymes and were designated as dual-carbapenemase producers, while one isolate (1.8%) was identified as a non-carbapenemase-producer. The CPO panel demonstrated positive and negative percent agreements of 100% and 85.7% for Ambler Class A, 100% and 100% for Class B, and 96.4% and 100% for Class D carbapenemase detection, respectively. CONCLUSION: While the CPO panel's phenotypic performance was satisfactory in detecting Class B and D carbapenemases, additional confirmatory testing may be necessary for Class A carbapenemases as part of routine laboratory procedures.


Assuntos
Proteínas de Bactérias , Infecções por Klebsiella , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , beta-Lactamases , beta-Lactamases/genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/efeitos dos fármacos , Proteínas de Bactérias/genética , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/diagnóstico , Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos
2.
PLoS One ; 19(5): e0303872, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38771780

RESUMO

BACKGROUND: Antimicrobial resistance (AMR) is among the top public health concerns in the globe. Estimating the prevalence of multidrug resistance (MDR), MDR index (MDR-I) and extended-spectrum beta-lactamase (ESBL)-producing lactose fermenting Enterobacteriaceae (LFE) is important in designing strategies to combat AMR. Thus, this study was designed to determine the status of MDR, MDR-I and ESBL-producing LFE isolated from the human-dairy interface in the northwestern part of Ethiopia, where such information is lacking. METHODOLOGY: A cross-sectional study was conducted from June 2022 to August 2023 by analyzing 362 samples consisting of raw pooled milk (58), milk container swabs (58), milker's hand swabs (58), farm sewage (57), milker's stool (47), and cow's feces (84). The samples were analyzed using standard bacteriological methods. The antimicrobial susceptibility patterns and ESBL production ability of the LFE isolates were screened using the Kirby-Bauer disk diffusion method, and candidate isolates passing the screening criteria were phenotypically confirmed by using cefotaxime (30 µg) and cefotaxime /clavulanic acid (30 µg/10 µg) combined-disk diffusion test. The isolates were further characterized genotypically using multiplex polymerase chain reaction targeting the three ESBL-encoding- genes namely blaTEM, blaSHV, and blaCTX-M. RESULTS: A total of 375 bacterial isolates were identified and the proportion of MDR and ESBL-producing bacterial isolates were 70.7 and 21.3%, respectively. The MDR-I varied from 0.0 to 0.81 with an average of 0.30. The ESBL production was detected in all sample types. Genotypically, the majority of the isolates (97.5%), which were positive on the phenotypic test, were carrying one or more of the three genes. CONCLUSION: A high proportion of the bacterial isolates were MDR; had high MDR-I and were positive for ESBL production. The findings provide evidence that the human-dairy interface is one of the important reservoirs of AMR traits. Therefore, the implementation of AMR mitigation strategies is highly needed in the area.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Enterobacteriaceae , Lactose , beta-Lactamases , Humanos , Etiópia , beta-Lactamases/genética , beta-Lactamases/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/enzimologia , Lactose/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Estudos Transversais , Antibacterianos/farmacologia , Animais , Testes de Sensibilidade Microbiana , Bovinos , Infecções por Enterobacteriaceae/microbiologia , Cefotaxima/farmacologia , Leite/microbiologia , Fermentação , Fezes/microbiologia
3.
Sci Rep ; 14(1): 10897, 2024 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740876

RESUMO

Urinary tract infection (UTI) is the most prevalent urological condition worldwide. Choosing appropriate antibiotics for patients who have fever before receiving a culture result is challenging. This retrospective study enrolled patients 394 patients hospitalized at Gangneung Asan Hospital for UTI from May 2017 to April 2021. Fever at 48 h of hospitalization was the analysis point, as this is when the response to antibiotic therapy manifest, although the results of antibiogram are not available. Multivariate analysis was performed to assess the correlation between ESBL producing bacteria (EPB) and fever at 48 h. Overall, 36.3% of patients had EPB and 27.9% had fever at 48 h. In multivariate analysis, a significant positive association was found between EPB and fever (odds ratio 1.17, 95% CI 1.05-1.30, P = 0.004) Female had negative association with multivariate model (OR 0.83, 95% CI 0.73-0.94, P = 0.004). Diabetes did not demonstrate a significant association with EPB. (OR 1.10, 95% CI 0.99-1.22, P = 0.072). Fever at 48 h is associated with EPB and could be considered a predictive factor for EPB infection in patients with UTI. Antibiotic escalation may be considered in patients with fever at 48 h.


Assuntos
Antibacterianos , Febre , Infecções Urinárias , beta-Lactamases , Humanos , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Feminino , Masculino , beta-Lactamases/metabolismo , Estudos Retrospectivos , Idoso , Pessoa de Meia-Idade , Antibacterianos/uso terapêutico , Antibacterianos/farmacologia , Febre/microbiologia , Febre/tratamento farmacológico , Idoso de 80 Anos ou mais , Adulto
4.
Curr Microbiol ; 81(7): 177, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38758473

RESUMO

The purpose of this study was to determine if orangutans (Pongo spp.) living in captivity at a zoo in Wisconsin were colonized with antimicrobial-resistant bacteria and, if found, to identify underlying genetic mechanisms contributing to their resistant phenotypes. We hypothesize that since antimicrobial-resistant bacteria are so prevalent within humans, the animals could also be carriers of such strains given the daily contact between the animals and the zoo staff that care for them. To test this theory, fecal samples from two orangutans were examined for resistant bacteria by inoculation on HardyCHROM™ ESBL and HardyCHROM™ CRE agars. Isolates were identified using MALDI-TOF mass spectrometry and antimicrobial susceptibility testing was performed using a Microscan autoSCAN-4 System. An isolate was selected for additional characterization, including whole genome sequencing (WGS). Using the Type (Strain) Genome Server (TYGS) the bacterium was identified as Escherichia coli. The sequence type identified was (ST/phylogenetic group/ß-lactamase): ST6448/B1/CTX-M-55.


Assuntos
Antibacterianos , Infecções por Escherichia coli , Escherichia coli , Fezes , beta-Lactamases , Animais , beta-Lactamases/genética , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Fezes/microbiologia , Antibacterianos/farmacologia , Animais de Zoológico/microbiologia , Testes de Sensibilidade Microbiana , Filogenia , Sequenciamento Completo do Genoma , Wisconsin , Proteínas de Escherichia coli/genética , Genoma Bacteriano
5.
Virulence ; 15(1): 2348251, 2024 12.
Artigo em Inglês | MEDLINE | ID: mdl-38697754

RESUMO

OBJECTIVES: This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). METHODS: Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. RESULTS: Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. CONCLUSIONS: In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.


Assuntos
Antibacterianos , Compostos Azabicíclicos , Proteínas de Bactérias , Ceftazidima , Combinação de Medicamentos , Infecções por Klebsiella , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana , Ceftazidima/farmacologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/patogenicidade , Klebsiella pneumoniae/enzimologia , Compostos Azabicíclicos/farmacologia , Antibacterianos/farmacologia , Masculino , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/tratamento farmacológico , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismo , Idoso , Farmacorresistência Bacteriana Múltipla/genética , Virulência , Plasmídeos/genética , Sequenciamento Completo do Genoma
6.
Sci Rep ; 14(1): 11260, 2024 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-38755240

RESUMO

Β-lactamases-producing Escherichia coli are a widely distributed source of antimicrobial resistance (AMR), for animals and humans. Little is known about the sensitivity profile and genetic characteristics of E. coli strains isolated from domestic cats. We report a cross-sectional study that evaluated E. coli strains isolated from domestic cats in Panama. For this study the following antibiotics were analyzed: ampicillin, amoxicillin-clavulanate cefepime, cefotaxime, cefoxitin, ceftazidime, aztreonam, imipenem, gentamicin, kanamycin, streptomycin, tetracycline, ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, and chloramphenicol. The data obtained were classified as resistant, intermediate, or sensitive. MDR strains were established when the strain presented resistance to at least one antibiotic from three or more antimicrobial classes. Forty-eight E. coli isolates were obtained, of which 80% presented resistance to at least one of the antibiotics analyzed, while only 20% were sensitive to all (p = 0.0001). The most common resistance was to gentamicin (58%). Twenty-nine percent were identified as multidrug-resistant isolates and 4% with extended spectrum beta-lactamase phenotype. The genes blaTEM (39%), blaMOX(16%), blaACC (16%) and blaEBC (8%) were detected. Plasmid-mediated resistance qnrB (25%) and qnrA (13%) are reported. The most frequent sequence types (STs) being ST399 and we reported 5 new STs. Our results suggest that in intestinal strains of E. coli isolated from domestic cats there is a high frequency of AMR.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Escherichia coli , Testes de Sensibilidade Microbiana , Animais , Gatos/microbiologia , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Antibacterianos/farmacologia , Fenótipo , beta-Lactamases/genética , Estudos Transversais , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Variação Genética
7.
PLoS One ; 19(5): e0303753, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38758757

RESUMO

NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice for these infections. However, there is still no recommendation of a practically feasible method for testing aztreonam and ceftazidime-avibactam synergy. We proposed a simple method for testing aztreonam and ceftazidime-avibactam synergy and compared it with reference broth micro-dilution and other methods. Carbapenem-resistant Enterobacterales clinical isolates were screened for the presence of the NDM gene by the Carba R test. NDM harbouring isolates were tested for aztreonam and ceftazidime-avibactam synergy by broth microdilution (reference method), E strip-disc diffusion, double disc diffusion, and disc replacement methods. In the newly proposed method, the MHA medium was supplemented with ceftazidime-avibactam (corresponding to an aztreonam concentration of 4µg/ml). The MHA medium was then inoculated with the standard inoculum (0.5 McFarland) of the test organism. An AZT disc (30 µg) was placed on the supplemented MHA medium, and the medium was incubated overnight at 37°C. Aztreonam zone diameter on the supplemented MHA medium (in the presence of ceftazidime-avibactam) was compared with that from a standard disc diffusion plate (without ceftazidime-avibactam), performed in parallel. Interpretation of synergy was based on the restoration of aztreonam zone diameter (in the presence of ceftazidime-avibactam) crossing the CLSI susceptibility breakpoint, i.e., ≥ 21 mm. Of 37 carbapenem-resistant NDM-producing isolates, 35 (94.6%) were resistant to aztreonam and tested synergy positive by the proposed method. Its sensitivity and specificity were 97.14% and 100%, respectively. Cohen's kappa value showed substantial agreement of the reference method with the proposed method (κ = 0.78) but no other methods. The proposed method is simple, easily interpretable, and showed excellent sensitivity, specificity, and agreement with the reference method. Therefore, the new method is feasible and reliable for testing aztreonam synergy with avibactam in NDM-producing Enterobacterales.


Assuntos
Antibacterianos , Compostos Azabicíclicos , Aztreonam , Ceftazidima , Combinação de Medicamentos , Enterobacteriaceae , Testes de Sensibilidade Microbiana , beta-Lactamases , Ceftazidima/farmacologia , Aztreonam/farmacologia , Compostos Azabicíclicos/farmacologia , beta-Lactamases/metabolismo , beta-Lactamases/genética , Testes de Sensibilidade Microbiana/métodos , Antibacterianos/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Humanos , Sinergismo Farmacológico , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/tratamento farmacológico
8.
Ann Clin Microbiol Antimicrob ; 23(1): 47, 2024 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-38796461

RESUMO

BACKGROUND: Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches. METHODS: 96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher). RESULTS: According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%). CONCLUSIONS: The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.


Assuntos
Antibacterianos , Compostos Azabicíclicos , Aztreonam , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas , Testes de Sensibilidade Microbiana , Aztreonam/farmacologia , Compostos Azabicíclicos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Antibacterianos/farmacologia , Humanos , Bactérias Gram-Negativas/efeitos dos fármacos , Combinação de Medicamentos , Pseudomonas aeruginosa/efeitos dos fármacos , beta-Lactamases/metabolismo , Enterobacteriaceae/efeitos dos fármacos , Proteínas de Bactérias , Infecções por Bactérias Gram-Negativas/microbiologia
9.
Ecotoxicol Environ Saf ; 278: 116395, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38728939

RESUMO

Escherichia coli (E. coli) plays an important ecological role, and is a useful bioindicator to recognize the evolution of resistance in human, animal and environment. Recently, extended-spectrum ß-lactamases (ESBL) producing E.coli has posed a threat to public health. Generally, captive healthy giant pandas are not exposed to antibiotics; however, they still acquire antimicrobial resistant bacteria. In order to understand whether there is an exchange of resistance genes within the ecosystems of captive giant pandas, this study explored resistance characteristics of 330 commensal E. coli isolates from feces of giant pandas, the surroundings, and breeders. Isolates from different sources showed similar resistance phenotype, and ESBL/AmpC-producing isolates showed more profound resistance to antibiotics than non-ESBL/AmpC-producing isolates (P<0.05). Furthermore, the occurrence of broad-spectrum ß-lactamase related resistance genes and colistin resistance genes was detected, and isolates phylogenetic typing and multilocus sequence typing (MLST) were applied in this study. Seven different ß-lactamase resistance genes (blaCTX-M-55, blaCTX-M-15, blaCTX-M-27, blaCTX-M-65, blaTEM-1, blaOXA-1 and blaCMY) and mcr-1 were found in 68 ESBL/AmpC-producing isolates. blaCTX-M-55 (48.53 %) was found the most predominant resistance genes, followed by blaTEM-1 (19.12 %) and blaCTX-M-27 (16.18 %). Nonetheless, blaCTX-M-55 was commonly detected in the isolates from giant pandas (63.16 %), the surroundings (43.48 %), and breeders (33.33 %). However, there were no carbapenemase genes detected in this study. mcr-1 was harbored in only one isolate from giant panda. Forty-five tansconjugants were successfully obtained in the conjugation experiments. The presence of antimicrobial resistance and related resistance genes tested were observed in the transconjugants. The results indicated that 52.63 % of the isolates from giant panda 73.91 % of the isolates from surroundings, and 100 % of the isolates from breeders were phylogroup A. Total of 27 sequence types (ST) were recognized from the isolate by MLST and found that ST48 (19/68; 27.94 %) was the predominant ST type, especially in the isolates from giant pandas and the surroundings. In conclusion, commensal ESBL/AmpC-producing E. coli becomes a reservoir of ESBL resistance genes, which is a potential threaten to health of giant pandas. The interaction between giant pandas, surroundings and breeders contribute to development of resistant phenotypes and genotypes which might transfer across species or the surroundings easily; hence, strict monitoring based on a "One Health" approach is recommended.


Assuntos
Antibacterianos , Proteínas de Bactérias , Escherichia coli , Fezes , Tipagem de Sequências Multilocus , Ursidae , beta-Lactamases , Animais , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , beta-Lactamases/genética , Ursidae/microbiologia , China , Antibacterianos/farmacologia , Fezes/microbiologia , Proteínas de Bactérias/genética , Ecossistema , Filogenia , Testes de Sensibilidade Microbiana , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Farmacorresistência Bacteriana Múltipla/genética , Farmacorresistência Bacteriana/genética
10.
BMC Genomics ; 25(1): 508, 2024 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-38778284

RESUMO

BACKGROUND: Enzymatic degradation mediated by beta-lactamases constitutes one of the primary mechanisms of resistance to beta-lactam antibiotics in gram-negative bacteria. This enzyme family comprises four molecular classes, categorized into serine beta-lactamases (Classes A, C, and D) and zinc-dependent metallo-beta-lactamases (Class B). Gram-negative bacteria producing beta-lactamase are of significant concern, particularly due to their prevalence in nosocomial infections. A comprehensive understanding of the evolution and dissemination of this enzyme family is essential for effective control of these pathogens. In this study, we conducted the prospecting, phylogenetic analysis, and in silico analysis of beta-lactamases and homologous proteins identified in 1827 bacterial genomes with phenotypic data on beta-lactam resistance. These genomes were distributed among Klebsiella pneumoniae (45%), Acinetobacter baumannii (31%), Pseudomonas aeruginosa (14%), Escherichia coli (6%), and Enterobacter spp. (4%). Using an HMM profile and searching for conserved domains, we mined 2514, 8733, 5424, and 2957 proteins for molecular classes A, B, C, and D, respectively. This set of proteins encompasses canonical subfamilies of beta-lactamases as well as hypothetical proteins and other functional groups. Canonical beta-lactamases were found to be phylogenetically distant from hypothetical proteins, which, in turn, are closer to other representatives of the penicillin-binding-protein (PBP-like) and metallo-beta-lactamase (MBL) families. The catalytic amino acid residues characteristic of beta-lactamases were identified from the sequence alignment and revealed that motifs are less conserved in homologous groups than in beta-lactamases. After comparing the frequency of protein groups in genomes of resistant strains with those of sensitive ones applying Fisher's exact test and relative risk, it was observed that some groups of homologous proteins to classes B and C are more common in the genomes of resistant strains, particularly to carbapenems. We identified the beta-lactamase-like domain widely distributed in gram-negative species of the ESKAPEE group, which highlights its importance in the context of beta-lactam resistance. Some hypothetical homologous proteins have been shown to potentially possess promiscuous activity against beta-lactam antibiotics, however, they do not appear to expressly determine the resistance phenotype. The selective pressure due to the widespread use of antibiotics may favor the optimization of these functions for specialized resistance enzymes.


Assuntos
Bactérias Gram-Negativas , Filogenia , beta-Lactamases , beta-Lactamases/metabolismo , beta-Lactamases/genética , beta-Lactamases/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , beta-Lactamas/farmacologia , beta-Lactamas/metabolismo , Antibacterianos/farmacologia , Genoma Bacteriano , Resistência beta-Lactâmica/genética , Antibióticos beta Lactam
11.
PLoS One ; 19(5): e0301531, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38787855

RESUMO

Wastewater discharge into the environment in resource-poor countries poses a threat to public health. Studies in this area within these countries are limited, and the use of high-throughput whole-genome sequencing technologies is lacking. Therefore, understanding of environmental impacts is inadequate. The present study investigated the antibiotic resistance profiles and diversity of beta-lactamases in Escherichia coli strains isolated from environmental water sources in Accra, Ghana. Microbiological analyses were conducted on wastewater samples from three hospitals, a sewage and wastewater treatment plant, and water samples from two urban surface water bodies. Confirmed isolates (N = 57) were selected for phenotypic antibiotic resistance profiles. Multi-drug-resistant isolates (n = 25) were genome sequenced using Illumina MiSeq sequencing technology and screened for sequence types, antibiotic resistance, virulence and beta-lactamase genes, and mobile genetic elements. Isolates were frequently resistant to ampicillin (63%), meropenem (47%), azithromycin (46%), and sulfamethoxazole-trimethoprim (42%). Twenty different sequence types (STs) were identified, including clinically relevant ones such as ST167 and ST21. Five isolates were assigned to novel STs: ST14531 (n = 2), ST14536, ST14537, and ST14538. The isolates belonged to phylogroups A (52%), B1 (44%), and B2 (4%) and carried ß-lactamase (TEM-1B, TEM-1C, CTX-M-15, and blaDHA-1) and carbapenemase (OXA-1, OXA-181) resistance genes. Dominant plasmid replicons included Col440I (10.2%) and IncFIB (AP001918) (6.8%). Polluted urban environments in Accra are reservoirs for antibiotic-resistant bacteria, posing a substantial public health risk. The findings underscore the need for targeted public health interventions to mitigate the spread of antibiotic-resistant bacteria and protect public health.


Assuntos
Farmacorresistência Bacteriana Múltipla , Escherichia coli , Águas Residuárias , beta-Lactamases , Gana , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , beta-Lactamases/genética , Humanos , Águas Residuárias/microbiologia , Saúde Pública , Antibacterianos/farmacologia , Microbiologia da Água , Testes de Sensibilidade Microbiana , Genômica , Sequenciamento Completo do Genoma , Filogenia , Esgotos/microbiologia , Genoma Bacteriano
12.
New Microbiol ; 47(1): 1-14, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38700878

RESUMO

Antibiotics are designed to effectively treat bacterial infections while minimizing harm to the human body. They work by targeting specific components of bacteria or by disrupting essential processes such as cell wall synthesis, membrane function, protein production, and metabolic pathways. However, the misuse and overuse of antibiotics have led to the emergence of drug resistance in humans, animals, and agriculture, contributing to the global spread of this problem. Drug resistance can be either innate or acquired, with acquired resistance involving changes in the bacterial chromosomes or transferable elements. Bacterial species employ various mechanisms of drug resistance, including modifying the antibiotic targets, inactivating the drug, reducing uptake or increasing efflux, overexpressing the target, utilizing alternative pathways, and forming biofilms. One significant concern in the realm of drug resistance revolves around the emergence and proliferation of extended-spectrum beta-lactamases (ESBLs), a gene that is found in most gram-negative bacteria, primarily carried by Escherichia coli and Klebsiella pneumoniae in healthcare settings. ESBL-mediated resistance poses challenges for diagnosis, treatment, infection control, and antibiotic stewardship. Accurate detection of ESBL genes is crucial, and phenotypic methods are commonly used for initial screening. However, these methods have limitations, and confirmatory molecular techniques such as PCR and DNA sequencing are employed to accurately identify ESBL genes. Despite the significant global concerns surrounding ESBLs, they have spread worldwide, mainly facilitated by healthcare settings, inappropriate antimicrobial use, and host susceptibility. Addressing this issue requires implementing comprehensive measures, including enhanced surveillance, strict infection control practices, antibiotic stewardship programs, rapid diagnostic methods, alternative therapies, public education initiatives, and research focused on developing new drugs. Furthermore, collaboration among the healthcare, public health, and research sectors is pivotal in effectively combating the escalating threat posed by ESBL-mediated resistance. Antibiotics have revolutionized medical care by effectively treating bacterial infections. However, the emergence of ESBL gene resistance poses a global challenge that requires an integrated approach to prevent a threatening future.


Assuntos
Antibacterianos , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Humanos , Animais , Farmacorresistência Bacteriana/genética , Fenótipo , Bactérias/efeitos dos fármacos , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
13.
Anal Methods ; 16(19): 3142-3153, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38713147

RESUMO

We develop and validate a method for the rapid determination and identification of 20 ß-lactamase antibiotics traces in goat's milk by combining the solid phase extraction technology with ultra-high performance liquid chromatography-tandem mass spectrometry. Goat milk samples were extracted with acetonitrile twice. The supernatant was then extracted and cleaned by solid-phase extraction using divinylbenzene and N-vinylpyrrolidone copolymer. The method was validated, with limits of quantification (LOQs) of 0.3 µg kg-1, specificities of 1/3 LOQ, linearities (R2) > 0.99, recoveries of 80-110%, repeatabilities <10.0%, and intermediate precisions <10.0%. The developed method was suitable for the routine analysis of ß-lactamase antibiotics residues in goat's milk and was used to test 76 goat milk samples produced in China.


Assuntos
Antibacterianos , Cabras , Leite , Extração em Fase Sólida , Espectrometria de Massas em Tandem , beta-Lactamases , Animais , Extração em Fase Sólida/métodos , Leite/química , Espectrometria de Massas em Tandem/métodos , Antibacterianos/análise , Cromatografia Líquida de Alta Pressão/métodos , Limite de Detecção , Reprodutibilidade dos Testes , Resíduos de Drogas/análise , Contaminação de Alimentos/análise , Espectrometria de Massa com Cromatografia Líquida
14.
Sci Rep ; 14(1): 11880, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38789462

RESUMO

Plasmids encoding blaCTX-M genes have greatly shaped the evolution of E. coli producing extended-spectrum beta-lactamases (ESBL-E. coli) and adds to the global threat of multiresistant bacteria by promoting horizontal gene transfer (HGT). Here we screened the similarity of 47 blaCTX-M -encoding plasmids, from 45 epidemiologically unrelated and disperse ESBL-E. coli strains, isolated during the early phase (2009-2014) of the ESBL pandemic in western Sweden. Using optical DNA mapping (ODM), both similar and rare plasmids were identified. As many as 57% of the plasmids formed five ODM-plasmid groups of at least three similar plasmids per group. The most prevalent type (28%, IncIl, pMLST37) encoded blaCTX-M-15 (n = 10), blaCTX-M-3 (n = 2) or blaCTX-M-55 (n = 1). It was found in isolates of various sequence types (STs), including ST131. This could indicate ongoing local HGT as whole-genome sequencing only revealed similarities with a rarely reported, IncIl plasmid. The second most prevalent type (IncFII/FIA/FIB, F1:A2:B20) harboring blaCTX-M-27, was detected in ST131-C1-M27 isolates, and was similar to plasmids previously reported for this subclade. The results also highlight the need for local surveillance of plasmids and the importance of temporospatial epidemiological links so that detection of a prevalent plasmid is not overestimated as a potential plasmid transmission event in outbreak investigations.


Assuntos
Escherichia coli , Plasmídeos , beta-Lactamases , Suécia/epidemiologia , Plasmídeos/genética , beta-Lactamases/genética , Escherichia coli/genética , Humanos , Pandemias , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Transferência Genética Horizontal , Proteínas de Escherichia coli/genética , Sequenciamento Completo do Genoma
15.
Ann Clin Microbiol Antimicrob ; 23(1): 46, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38790053

RESUMO

BACKGROUND: Proteus mirabilis is an opportunistic pathogen that has been held responsible for numerous nosocomial and community-acquired infections which are difficult to be controlled because of its diverse antimicrobial resistance mechanisms. METHODS: Antimicrobial susceptibility patterns of P. mirabilis isolates collected from different clinical sources in Mansoura University Hospitals, Egypt was determined. Moreover, the underlying resistance mechanisms and genetic relatedness between isolates were investigated. RESULTS: Antimicrobial susceptibility testing indicated elevated levels of resistance to different classes of antimicrobials among the tested P. mirabilis clinical isolates (n = 66). ERIC-PCR showed great diversity among the tested isolates. Six isolates (9.1%) were XDR while all the remaining isolates were MDR. ESBLs and AmpCs were detected in 57.6% and 21.2% of the isolates, respectively, where blaTEM, blaSHV, blaCTX-M, blaCIT-M and blaAmpC were detected. Carbapenemases and MBLs were detected in 10.6 and 9.1% of the isolates, respectively, where blaOXA-48 and blaNDM-1 genes were detected. Quinolone resistant isolates (75.8%) harbored acc(6')-Ib-cr, qnrD, qnrA, and qnrS genes. Resistance to aminoglycosides, trimethoprim-sulfamethoxazole and chloramphenicol exceeded 80%. Fosfomycin was the most active drug against the tested isolates as only 22.7% were resistant. Class I or II integrons were detected in 86.4% of the isolates. Among class I integron positive isolates, four different gene cassette arrays (dfrA17- aadA5, aadB-aadA2, aadA2-lnuF, and dfrA14-arr-3-blaOXA-10-aadA15) and two gene cassettes (dfrA7 and aadA1) were detected. While class II integron positive isolates carried four different gene cassette arrays (dfrA1-sat1-aadA1, estXVr-sat2-aadA1, lnuF- dfrA1-aadA1, and dfrA1-sat2). CONCLUSION: P. Mirabilis ability to acquire resistance determinants via integrons may be held responsible for the elevated rates of antimicrobial resistance and emergence of XDR or even PDR strains limiting the available therapeutic options for management of infections caused by those strains.


Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Infecções por Proteus , Proteus mirabilis , Egito/epidemiologia , Humanos , Proteus mirabilis/genética , Proteus mirabilis/efeitos dos fármacos , Proteus mirabilis/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Proteus/microbiologia , Infecções por Proteus/epidemiologia , Antibacterianos/farmacologia , Prevalência , beta-Lactamases/genética , Integrons/genética , Proteínas de Bactérias/genética , Infecção Hospitalar/microbiologia , Infecção Hospitalar/epidemiologia , Masculino
16.
J Microbiol Methods ; 221: 106940, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38702032

RESUMO

Bloodstream infections (BSI) caused by carbapenem-resistant Gram-negative bacilli (CR-GNB) are a subject of major clinical concern, mainly those associated with carbapenemase-producing isolates. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed to detect specific ß-lactamases, including KPC. We aimed to detect KPC enzyme directly from positive blood cultures using MALDI-TOF MS. Overall, 146 clinical Gram-negative bacilli (46 CR-GNB) recovered from consecutive blood cultures were evaluated. Proteins were extracted using formic acid, isopropyl alcohol, and water and spotted onto a steel target plate using the double-layer sinapinic acid method. The relative ions intensity ≥120 arbitrary units (a.u.) of a peak close to 28,700 m/z indicated the presence of KPC. The results were compared to HRM-qPCR methodology. This specific peak was observed in 11/14 blood bottles with blaKPC positive isolates (78.6% sensitivity), with 3 false-positive results (97.7% specificity). Analysis from colonies reached identical sensitivity (78.6%), but higher specificity (100%). The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid. It's excellent specificity indicates that positive results are consistently associated with the presence of a KPC producer in positive blood culture.


Assuntos
Proteínas de Bactérias , Hemocultura , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Lactamases , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Humanos , beta-Lactamases/genética , Hemocultura/métodos , Proteínas de Bactérias/genética , Sensibilidade e Especificidade , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Bacteriemia/microbiologia , Bacteriemia/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/sangue , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia
17.
PLoS One ; 19(5): e0303353, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38743684

RESUMO

INTRODUCTION: The study of Klebsiella quasipneumoniae, Klebsiella variicola, and AmpC production in extended-spectrum ß-lactamase (ESBL)-producing Klebsiella in Japan is limited, and existing data are insufficient. This study aims to characterize Klebsiella species, determine AmpC production rates, and analyze antimicrobial resistance patterns in ESBL-producing Klebsiella isolates in Japan. METHODS: A total of 139 clinical isolates of ESBL-producing Klebsiella were collected in Japan, along with their corresponding antimicrobial susceptibility profiles. The isolates were identified using a web-based tool. ESBL genes within the isolates were identified using multiplex PCR. Screening for AmpC-producing isolates was performed using cefoxitin disks, followed by multiplex PCR to detect the presence of AmpC genes. Antimicrobial resistance patterns were analyzed across the predominant ESBL genotypes. RESULTS: The web-based tool identified 135 isolates (97.1%) as Klebsiella pneumoniae and 4 (2.9%) as K. quasipneumoniae subsp. similipneumoniae, with no instances of K. variicola detected. Among K. pneumoniae, the CTX-M-1 group emerged as the predominant genotype (83/135, 61.5%), followed by K. quasipneumoniae subsp. similipneumoniae (3/4, 75.0%). The CTX-M-9 group was the second most prevalent genotype in K. pneumoniae (45/135, 33.3%). The high resistance rates were observed for quinolones (ranging from 46.7% to 63.0%) and trimethoprim/sulfamethoxazole (78.5%). The CTX-M-1 group exhibited higher resistance to ciprofloxacin (66/83, 79.5%) compared to the CTX-M-9 group (18/45, 40.0%), a trend also observed for levofloxacin and trimethoprim/sulfamethoxazole. Among the 16 isolates that tested positive during AmpC screening, only one K. pneumoniae isolates (0.7%) were confirmed to carry the AmpC gene. CONCLUSION: Klebsiella pneumoniae with the CTX-M-1 group is the most common ESBL-producing Klebsiella in Japan and showed a low proportion of AmpC production. These isolates are resistant to quinolones and trimethoprim/sulfamethoxazole, highlighting the challenge of managing this pathogen. The findings underscore the importance of broader research and continuous monitoring to address the resistance patterns of ESBL-producing Klebsiella.


Assuntos
Antibacterianos , Proteínas de Bactérias , Infecções por Klebsiella , Klebsiella pneumoniae , Klebsiella , Testes de Sensibilidade Microbiana , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Klebsiella pneumoniae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Klebsiella/genética , Klebsiella/efeitos dos fármacos , Klebsiella/isolamento & purificação , Klebsiella/enzimologia , Japão , Estudos Retrospectivos , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/epidemiologia , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Masculino , Feminino , População do Leste Asiático
18.
Nat Commun ; 15(1): 4093, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38750030

RESUMO

Plasmids carrying antibiotic resistance genes (ARG) are the main mechanism of resistance dissemination in Enterobacterales. However, the fitness-resistance trade-off may result in their elimination. Chromosomal integration of ARGs preserves resistance advantage while relieving the selective pressure for keeping costly plasmids. In some bacterial lineages, such as carbapenemase producing sequence type ST38 Escherichia coli, most ARGs are chromosomally integrated. Here we reproduce by experimental evolution the mobilisation of the carbapenemase blaOXA-48 gene from the pOXA-48 plasmid into the chromosome. We demonstrate that this integration depends on a plasmid-induced fitness cost, a mobile genetic structure embedding the ARG and a novel antiplasmid system ApsAB actively involved in pOXA-48 destabilization. We show that ApsAB targets high and low-copy number plasmids. ApsAB combines a nuclease/helicase protein and a novel type of Argonaute-like protein. It belongs to a family of defense systems broadly distributed among bacteria, which might have a strong ecological impact on plasmid diffusion.


Assuntos
Escherichia coli , Plasmídeos , beta-Lactamases , Plasmídeos/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Cromossomos Bacterianos/genética
19.
Biochemistry ; 63(10): 1278-1286, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38690676

RESUMO

Metallo-ß-lactamases (MBL) deactivate ß-lactam antibiotics through a catalytic reaction caused by two zinc ions at the active center. Since MBLs deteriorate a wide range of antibiotics, they are dangerous factors for bacterial multidrug resistance. In this work, organic synthesis, computational design, and crystal structure analysis were performed to obtain potent MBL inhibitors based on a previously identified hit compound. The hit compound comprised 3,4-dihydro-2(1H)-quinolinone linked with a phenyl-ether-methyl group via a thiazole ring. In the first step, the thiazole ring was replaced with a tertiary amine to avoid the planar structure. In the second step, we virtually modified the compound by keeping the quinolinone backbone. Every modified compound was bound to a kind of MBL, imipenemase-1 (IMP-1), and the binding pose was optimized by a molecular mechanics calculation. The binding scores were evaluated for the respective optimized binding poses. Given the predicted binding poses and calculated binding scores, candidate compounds were determined for organic syntheses. The inhibitory activities of the synthesized compounds were measured by an in vitro assay for two kinds of MBLs, IMP-1 and New Delhi metallo-ß-lactamase (NDM-1). A quinolinone connected with an amine bound with methyl-phenyl-ether-propyl and cyclohexyl-ethyl showed a 50% inhibitory concentration of 4.8 µM. An X-ray crystal analysis clarified the binding structure of a synthesized compound to IMP-1. The δ-lactam ring of quinolinone was hydrolyzed, and the generated carboxyl group was coordinated with zinc ions. The findings on the chemical structure and binding pose are expected to be a base for developing MBL inhibitors.


Assuntos
Inibidores de beta-Lactamases , beta-Lactamases , beta-Lactamases/química , beta-Lactamases/metabolismo , Inibidores de beta-Lactamases/farmacologia , Inibidores de beta-Lactamases/química , Cristalografia por Raios X , Desenho de Fármacos , Simulação de Acoplamento Molecular , Antibacterianos/farmacologia , Antibacterianos/química , Quinolonas/química , Quinolonas/farmacologia , Quinolonas/metabolismo
20.
Sci Total Environ ; 931: 172873, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38692330

RESUMO

Carbapenem resistance's global proliferation poses a significant public health challenge. The primary resistance mechanism is carbapenemase production. In this study, we discovered a novel carbapenemase, RATA, located on the chromosome of Riemerella anatipestifer isolates. This enzyme shares ≤52 % amino acid sequence identity with other known ß-lactamases. Antimicrobial susceptibility tests and kinetic assays demonstrated that RATA could hydrolyze not only penicillins and extended-spectrum cephalosporins but also monobactams, cephamycins, and carbapenems. Furthermore, its activity was readily inhibited by ß-lactamase inhibitors. Bioinformatic analysis revealed 46 blaRATA-like genes encoding 27 variants in the NCBI database, involving 21 different species, including pathogens, host-associated bacteria, and environmental isolates. Notably, blaRATA-positive strains were globally distributed and primarily collected from marine environments. Concurrently, taxonomic analysis and GC content analysis indicated that blaRATA orthologue genes were predominantly located on the chromosomes of Flavobacteriaceae and shared a similar GC content as Flavobacteriaceae. Although no explicit mobile genetic elements were identified by genetic environment analysis, blaRATA-2 possessed the ability of horizontal transfer in R. anatipestifer via natural transformation. This work's data suggest that RATA is a new chromosome-encoded class A carbapenemase, and Flavobacteriaceae from marine environments could be the primary reservoir of the blaRATA gene.


Assuntos
Proteínas de Bactérias , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Carbapenêmicos/farmacologia
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