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1.
Nat Commun ; 11(1): 4915, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004811

RESUMO

A phenotype of Escherichia coli and Klebsiella pneumoniae, resistant to piperacillin/tazobactam (TZP) but susceptible to carbapenems and 3rd generation cephalosporins, has emerged. The resistance mechanism associated with this phenotype has been identified as hyperproduction of the ß-lactamase TEM. However, the mechanism of hyperproduction due to gene amplification is not well understood. Here, we report a mechanism of gene amplification due to a translocatable unit (TU) excising from an IS26-flanked pseudo-compound transposon, PTn6762, which harbours blaTEM-1B. The TU re-inserts into the chromosome adjacent to IS26 and forms a tandem array of TUs, which increases the copy number of blaTEM-1B, leading to TEM-1B hyperproduction and TZP resistance. Despite a significant increase in blaTEM-1B copy number, the TZP-resistant isolate does not incur a fitness cost compared to the TZP-susceptible ancestor. This mechanism of amplification of blaTEM-1B is an important consideration when using genomic data to predict susceptibility to TZP.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/tratamento farmacológico , Proteínas de Escherichia coli/genética , Escherichia coli/genética , beta-Lactamases/genética , Antibacterianos/uso terapêutico , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Quimioterapia Combinada/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Amplificação de Genes , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana , Piperacilina/farmacologia , Piperacilina/uso terapêutico , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Tazobactam/farmacologia , Tazobactam/uso terapêutico , Sequenciamento Completo do Genoma
2.
BMC Infect Dis ; 20(1): 673, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938424

RESUMO

BACKGROUND: Urinary tract infection is one of the most common bacterial infections in children. Understanding the characteristics of uropathogens and their antimicrobial susceptibility pattern in a particular setting can provide evidence for the appropriate management of cases. This study aimed to assess the bacterial profile of urinary tract infection, their antimicrobial susceptibility pattern and associated factors among clinically suspected children attending at Felege-Hiwot Comprehensive Specialized Hospital, Northwest Ethiopia. METHODS: A hospital-based cross-sectional study was conducted from February-April, 2019. A systematic sampling technique was employed. A mid-stream urine sample was inoculated on cystine lactose electrolyte deficient media and incubated for 24-48 h. Sub-culturing was done on Mac-Conkey and blood agar. Antimicrobial susceptibility test was done on Muller-Hinton agar. A binary logistic regression model was used to see the association between dependent and independent factors. A p-value< 0.05 at 95% CI was considered as statistically significant. RESULTS: The overall prevalence of urinary tract infection was 16.7% (95% CI 12.4-21.1). Both Gram-negative and Gram-positive bacterial isolates were recovered with a rate of 44/50 (88%) and 6/50 (12%) respectively. Among Gram-negative isolates, E. coli 28/44(63.6%) was predominant while S. saprophyticus 2/6(33.3%) was prevalent among Gram-positive bacterial isolates. Overall, a high level of resistance to ampicillin, augmentin, and tetracycline was shown by Gram-negative bacteria with a rate of 44/44(100%), 39/44(88.6%), and36/44 (81.8%) respectively. About 33/50(66%) of overall multidrug resistance was observed (95% CI 52-78). About six Gram-negative bacterial isolates were extended spectrum beta-lactamase (ESBL) producers. Having a history of urinary tract infection (P-0.003, AOR 1.86-22.15) and male uncircumcision (p-0.00, AOR 5.5-65.35) were the independent variables that associate for urinary tract infections. CONCLUSION: In the present study, the prevalence of urinary tract infection among children was high and considerably a high proportion of multidrug resistance was observed. This result will have a significant impact on the selection of appropriate antimicrobial agents for the treatment of urinary tract infection.


Assuntos
Infecções Urinárias/diagnóstico , Adolescente , Antibacterianos/farmacologia , Criança , Pré-Escolar , Estudos Transversais , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Etiópia/epidemiologia , Feminino , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/isolamento & purificação , Hospitais , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Razão de Chances , Estudos Prospectivos , Infecções Urinárias/microbiologia , beta-Lactamases/genética , beta-Lactamases/metabolismo
3.
BMC Infect Dis ; 20(1): 703, 2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32977759

RESUMO

BACKGROUND: Treatment of gonorrhea is complicated by the development of antimicrobial resistance in Neisseria gonorrhoeae (GC) to the antibiotics recommended for treatment. Knowledge on types of plasmids and the antibiotic resistance genes they harbor is useful in monitoring the emergence and spread of bacterial antibiotic resistance. In Kenya, studies on gonococcal antimicrobial resistance are few and data on plasmid mediated drug resistance is limited. The present study characterizes plasmid mediated resistance in N. gonorrhoeae isolates recovered from Kenya between 2013 and 2018. METHODS: DNA was extracted from 36 sub-cultured GC isolates exhibiting varying drug resistance profiles. Whole genome sequencing was done on Illumina MiSeq platform and reads assembled de-novo using CLC Genomics Workbench. Genome annotation was performed using Rapid Annotation Subsystem Technology. Comparisons in identified antimicrobial resistance determinants were done using Bioedit sequence alignment editor. RESULTS: Twenty-four (66.7%) isolates had both ß-lactamase (TEM) and TetM encoding plasmids. 8.3% of the isolates lacked both TEM and TetM plasmids and had intermediate to susceptible penicillin and tetracycline MICs. Twenty-six (72%) isolates harbored TEM encoding plasmids. 25 of the TEM plasmids were of African type while one was an Asian type. Of the 36 isolates, 31 (86.1%) had TetM encoding plasmids, 30 of which harbored American TetM, whereas 1 carried a Dutch TetM. All analyzed isolates had non-mosaic penA alleles. All the isolates expressing TetM were tetracycline resistant (MIC> 1 mg/L) and had increased doxycycline MICs (up to 96 mg/L). All the isolates had S10 ribosomal protein V57M amino acid substitution associated with tetracycline resistance. No relation was observed between PenB and MtrR alterations and penicillin and tetracycline MICs. CONCLUSION: High-level gonococcal penicillin and tetracycline resistance in the sampled Kenyan regions was found to be mediated by plasmid borne blaTEM and tetM genes. While the African TEM plasmid, TEM1 and American TetM are the dominant genotypes, Asian TEM plasmid, a new TEM239 and Dutch TetM have emerged in the regions.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/genética , Gonorreia/tratamento farmacológico , Gonorreia/epidemiologia , Neisseria gonorrhoeae/genética , Penicilinas/uso terapêutico , Plasmídeos/genética , Resistência a Tetraciclina/genética , Tetraciclina/uso terapêutico , DNA Bacteriano/genética , Feminino , Genótipo , Gonorreia/microbiologia , Humanos , Quênia/epidemiologia , Masculino , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/isolamento & purificação , Sequenciamento Completo do Genoma , beta-Lactamases/genética
4.
PLoS One ; 15(8): e0237474, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857767

RESUMO

The effective treatment of carbapenemase-producing Klebsiella pneumoniae infection has been limited and required novel potential agents. Due to the novel drug development crisis, using old antimicrobial agents and combination therapy have been highlighted. This study focused on fosfomycin which inhibits cell wall synthesis and has potential activity on Enterobacteriaceae. We evaluated fosfomycin activity against carbapenemase-producing K. pneumoniae and characterized fosfomycin resistance mechanisms. Fosfomycin revealed effective activity against only 31.8% of carbapenemase-producing K. pneumoniae isolates. The major resistance mechanism was FosA3 production. The co-occurrence of FosA3 overexpression with the mutation of glpT (or loss of glpT) and/or uhpT was mediated high-level resistance (MIC>256 mg/L) to fosfomycin. Moreover, fosA3 silenced in sixteen fosfomycin-susceptible isolates and the plasmid carrying fosA3 of these isolates increased 32- to 64-fold of fosfomycin MICs in Escherichia coli DH5α transformants. The in vitro activity of fosfomycin combination with amikacin by checkerboard assay showed synergism and no interaction in six (16.2%) and sixteen isolates (43.3%), respectively. No antagonism of fosfomycin and amikacin was observed. Notably, the silence of aac (6)'-Ib and aphA6 was observed in amikacin-susceptible isolates. Our study suggests that the combination of fosfomycin and amikacin may be insufficient for the treatment of carbapenemase-producing K. pneumoniae isolates.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Fosfomicina/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , beta-Lactamases/metabolismo , Amicacina/farmacologia , Substituição de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Escherichia coli/metabolismo , Humanos , Infecções por Klebsiella/microbiologia , Infecções por Klebsiella/patologia , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , beta-Lactamases/genética
5.
Sci Total Environ ; 746: 140894, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32763594

RESUMO

Multidrug-resistant bacteria cause difficult-to-treat infections and pose a risk for modern medicine. Sources of multidrug-resistant bacteria include hospital, municipal and slaughterhouse wastewaters. In this study, bacteria with resistance to 3rd generation cephalosporins were isolated from all three wastewater biotopes, including a maximum care hospital, municipal wastewaters collected separately from a city and small rural towns and the wastewaters of two pig and two poultry slaughterhouses. The resistance profiles of all isolates against clinically relevant antibiotics (including ß-lactams like carbapenems, the quinolone ciprofloxacin, colistin, and trimethoprim/sulfamethoxazole) were determined at the same laboratory. The bacteria were classified according to their risk to human health using clinical criteria, with an emphasis on producers of carbapenemases, since carbapenems are prescribed for hospitalized patients with infections with multi-drug resistant bacteria. The results showed that bacteria that pose the highest risk, i. e., bacteria resistant to all ß-lactams including carbapenems and ciprofloxacin, were mainly disseminated by hospitals and were present only in low amounts in municipal wastewater. The isolates from hospital wastewater also showed the highest rates of resistance against antibiotics used for treatment of carbapenemase producers and some isolates were susceptible to only one antibiotic substance. In accordance with these results, qPCR of resistance genes showed that 90% of the daily load of carbapenemase genes entering the municipal wastewater treatment plant was supplied by the clinically influenced wastewater, which constituted approximately 6% of the wastewater at this sampling point. Likewise, the signature of the clinical wastewater was still visible in the resistance profiles of the bacteria isolated at the entry into the wastewater treatment plant. Carbapenemase producers were not detected in slaughterhouse wastewater, but strains harboring the colistin resistance gene mcr-1 could be isolated. Resistances against orally available antibiotics like ciprofloxacin and trimethoprim/sulfamethoxazole were widespread in strains from all three wastewaters.


Assuntos
Matadouros , Águas Residuárias , Animais , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Carbapenêmicos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Hospitais Municipais , Humanos , Testes de Sensibilidade Microbiana , Suínos , beta-Lactamases/genética
6.
BMC Infect Dis ; 20(1): 635, 2020 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-32847524

RESUMO

BACKGROUND: Data regarding the prevalence of metallo-ß-lactamases (MBLs) among Pseudomonas aeruginosa isolates in cystic fibrosis patients are scarce. Furthermore, there is limited knowledge on the effect of MBL production on patient outcomes. Here we describe a fatal respiratory infection due to P. aeruginosa producing VIM-type MBLs in a lung transplant recipient and the results of the subsequent epidemiological investigation. CASE PRESENTATION: P. aeruginosa isolates collected in the index patient and among patients temporally or spatially linked with the index patient were analyzed in terms of antibiotic susceptibility profile and MBL production. Whole-genome sequencing and phylogenetic reconstruction were also performed for all P. aeruginosa isolates producing VIM-type MBLs. A VIM-producing P. aeruginosa strain was identified in a lung biopsy of a lung transplant recipient with cystic fibrosis. The strain was VIM-1-producer and belonged to the ST308. Despite aggressive treatment, the transplant patient succumbed to the pulmonary infection due to the ST308 strain. A VIM-producing P. aeruginosa strain was also collected from the respiratory samples of a different cystic fibrosis patient attending the same cystic fibrosis center. This isolate harbored the blaVIM-2 gene and belonged to the clone ST175. This patient did not experience an adverse outcome. CONCLUSIONS: This is the first description of a fatal infection due to P. aeruginosa producing VIM-type MBLs in a lung transplant recipient. The circulation of P. aeruginosa isolates harboring MBLs pose a substantial risk to the cystic fibrosis population due to the limited therapeutic options available and their spreading potential.


Assuntos
Antibacterianos/uso terapêutico , Transplante de Pulmão , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/enzimologia , Infecções Respiratórias/tratamento farmacológico , Transplantados , Adulto , Fibrose Cística/cirurgia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Evolução Fatal , Feminino , Humanos , Pulmão/microbiologia , Pulmão/patologia , Testes de Sensibilidade Microbiana , Filogenia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Infecções Respiratórias/microbiologia , beta-Lactamases/genética , beta-Lactamases/metabolismo
7.
PLoS One ; 15(7): e0235853, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701967

RESUMO

PCR-based amplification of annotated genes has allowed construction of expression clones at genome-scale using classical and recombination-based cloning technologies. However, genome-scale expression and purification of proteins for down-stream applications is often limited by challenges such as poor expression, low solubility, large size of multi-domain proteins, etc. Alternatively, DNA fragment libraries in expression vectors can serve as the source of protein fragments with each fragment encompassing a function of its whole protein counterpart. However, the random DNA fragmentation and cloning result in only 1 out of 18 clones being in the correct open-reading frame (ORF), thus, reducing the overall efficiency of the system. This necessitates the selection of correct ORF before expressing the protein fragments. This paper describes a highly efficient ORF selection system for DNA fragment libraries, which is based on split beta-lactamase protein fragment complementation. The system has been designed to allow seamless transfer of selected DNA fragment libraries into any downstream vector systems using a restriction enzyme-free cloning strategy. The strategy has been applied for the selection of ORF using model constructs to show near 100% selection of the clone encoding correct ORF. The system has been further validated by construction of an ORF-selected DNA fragment library of 30 genes of M. tuberculosis. Further, we have successfully demonstrated the cytosolic expression of ORF-selected protein fragments in E. coli.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Teste de Complementação Genética/métodos , Fases de Leitura Aberta , beta-Lactamases/genética , Proteínas de Bactérias/metabolismo , Escherichia coli , Biblioteca Gênica , Mycobacterium tuberculosis , beta-Lactamases/metabolismo
8.
J Med Microbiol ; 69(8): 1089-1094, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32692646

RESUMO

Introduction. The bla CTX-M-3 gene has rarely been reported in Morganella morganii strains and its genetic environment has not yet been investigated.Aim. To identify the bla CTX-M-3 gene in M. morganii isolated from swine and characterize its genetic environment.Methodology. A M. morganii isolate (named MM1L5) from a deceased swine was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing. The bla genes were detected and then the genetic location and environment of bla CTX-M-3 were investigated by Southern blot and PCR mapping, respectively. The M. morganii bla CTX-M-3 gene was cloned and expressed in Escherichia coli.Results. Isolate MM1L5 harboured the bla CTX-M-3 and bla TEM-1 genes. The bla CTX-M-3 gene, located on the chromosome, was co-carried with an IS26 and bla TEM-1 gene by a novel 6361 bp IS26-flanked composite transposon, designated Tn6741. This transposon consisted of a novel bla CTX-M-3-containing module, IS26-ΔISEcp1-bla CTX-M-3-Δorf477-IS26 (named Tn6710), and a bla TEM-1-containing module, IS26-Δorf477-bla TEM-1-tnpR-IS26, differing from previous reports. Phylogenetic analysis showed a significant variation based on the sequence of Tn6741, as compared to those of other related transposons. Interestingly, although the cloned bla CTX-M-3 gene could confer resistance to ceftiofur, cefquinome, ceftriaxone and cefotaxime, one amino acid substitution (Ile-142-Thr) resulted in a significant reduction of resistance to these antimicrobials.Conclusion. This is the first time that bla CTX-M-3 has been identified on a chromosome from a M. morganii isolate. Furthermore, the bla CTX-M-3 gene was located with an IS26 element and bla TEM-1 gene on a novel IS26-flanked composite transposon, Tn6741, suggesting that Tn6741 might act as a reservoir for the bla CTX-M-3 and bla TEM-1 genes and may become an important vehicle for their dissemination among M. morganii.


Assuntos
Elementos de DNA Transponíveis/genética , Morganella morganii/genética , beta-Lactamases/genética , Animais , Anti-Infecciosos/farmacologia , Sequência de Bases , Clonagem Molecular , Morganella morganii/classificação , Morganella morganii/efeitos dos fármacos , Filogenia , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos
9.
BMC Infect Dis ; 20(1): 544, 2020 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-32711470

RESUMO

BACKGROUND: This study aimed to identify ten different 16S rRNA methyltransferase genes (rmtA, rmtB, rmtC, rmtD, armA, rmtF, npmA, rmtH, rmtE and rmtG) and their coexisting ESBL and carbapenemase with the emergence of three E.coli clones within a single study centre. METHODS: A total of 329 non-duplicate E.coli isolates were studied to detect the presence of 16S rRNA methyltransferases along with ß-lactamases (TEM, SHV, OXA, VEB, GES, PER,CTX-M types, NDM, OXA-48,VIM, IMP and KPC) using PCR assay. Horizontal transferability were validated by transformation and conjugation analysis. Plasmid incompatibility typing and MLST analysis was also performed. RESULTS: A total of 117 isolates were found to be resistant to at least one of the aminoglycoside antibiotics. It was observed that 77 (65.8%) were positive for 16S rRNA methyltransferases. Among them thirty nine isolates were found to harbour only blaCTX-M-15, whereas combination of genes were observed in three isolates (blaVEB+ blaCTX-M-15 in 2 isolates and blaPER + blaCTX-M-15 in 1 isolate). blaNDM and blaOXA-48 like genes were found in 23 and 9 isolates, respectively. All the resistance genes were conjugatively transferable, and incompatibility typing showed multiple 16S rRNA methyltransferase genes were originated from a single Inc. I1 group. MLST analysis detected 3 clones of E.coliST4410, ST1341 and ST3906. CONCLUSION: The present study identified emergence of three clones of E.coli, resistant to aminoglycoside -cephalosporin- carbapenem. This warrants immediate measures to trace their transmission dynamics in order to slow down their spread in clinical setting.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Metiltransferases/genética , beta-Lactamases/genética , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/classificação , Escherichia coli/efeitos dos fármacos , Genes Bacterianos/genética , Humanos , Índia , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus
10.
BMC Infect Dis ; 20(1): 540, 2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32703276

RESUMO

BACKGROUND: Antimicrobial resistance is an ecological and multicausal problem. Infections caused by extended-spectrum ß-lactamase producing Enterobacteriaceae (ESBL-E) can be acquired and transmitted in the community. Data on community-associated ESBL-E infections/colonizations in Colombia are scarce. Georeferencing tools can be used to study the dynamics of antimicrobial resistance at the community level. METHODS: We conducted a study of geographic mapping using modern tools based on geographic information systems (GIS). Two study centers from the city of Pereira, Colombia were involved. The records of patients who had ESBL-producing Enterobacteriaceae were reviewed. Antimicrobial susceptibility testing and phenotypic detection of ESBL was done according to CLSI standards. RESULTS: A population of 415 patients with community-acquired infections/colonizations and 77 hospital discharges were obtained. Geographic distribution was established and heat maps were created. Several hotspots were evidenced in some geographical areas of the south-west and north-east of the city. Many of the affected areas were near tertiary hospitals, rivers, and poultry industry areas. CONCLUSIONS: There are foci of antimicrobial resistance at the community level. This was demonstrated in the case of antimicrobial resistance caused by ESBL in a city in Colombia. Causality with tertiary hospitals in the city, some rivers and the poultry industry is proposed as an explanation of the evidenced phenomenon. Geographic mapping tools are useful for monitoring antimicrobial resistance in the community.


Assuntos
Infecções Comunitárias Adquiridas/epidemiologia , Infecções por Enterobacteriaceae/epidemiologia , Enterobacteriaceae/enzimologia , Mapeamento Geográfico , Fenótipo , beta-Lactamases/genética , Adolescente , Adulto , Idoso , Antibacterianos/uso terapêutico , Colômbia/epidemiologia , Infecções Comunitárias Adquiridas/microbiologia , Farmacorresistência Bacteriana/genética , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/microbiologia , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Prevalência , Adulto Jovem
11.
PLoS One ; 15(7): e0236505, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701970

RESUMO

Multidrug resistance prompts the search for new sources of antibiotics with new targets at bacteria cell. To investigate the antibacterial activity of Cinnamomum cassia L. essential oil (CCeo) alone and in combination with antibiotics against carbapenemase-producing Klebsiella pneumoniae and Serratia marcescens. The antimicrobial susceptibility of the strains was determined by Vitek® 2 and confirmed by MALDI-TOF/TOF. The antibacterial activity of CCeo and its synergism with antibiotics was determined using agar disk diffusion, broth microdilution, time-kill, and checkboard methods. The integrity of the bacterial cell membrane in S. marcescens was monitored by protein leakage assay. CCeo exhibited inhibitory effects with MIC = 281.25 µg.mL-1. The association between CCeo and polymyxin B showed a decrease in terms of viable cell counts on survival curves over time after a 4 hour-treatment with a FIC index value of 0.006. Protein leakage was observed with increasing concentrations for CCeo and CCeo + polymyxin B treatments. CCeo showed antibacterial activity against the studied strains. When associated with polymyxin B, a synergistic effect was able to inhibit bacterial growth rapidly and consistently, making it a potential candidate for the development of an alternative treatment and drug delivery system for carbapenemase-producing strains.


Assuntos
Infecções por Klebsiella/tratamento farmacológico , Óleos Voláteis/farmacologia , Polimixina B/farmacologia , Infecções por Serratia/tratamento farmacológico , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Cinnamomum aromaticum/química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Sinergismo Farmacológico , Humanos , Infecções por Klebsiella/genética , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/patogenicidade , Infecções por Serratia/genética , Infecções por Serratia/microbiologia , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/patogenicidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Lactamases/genética
12.
PLoS One ; 15(7): e0234684, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702006

RESUMO

OBJECTIVE: To describe the clinical features, outcomes, and molecular epidemiology of an outbreak of multidrug resistant (MDR) A. baumannii. METHODS: We performed a retrospective analysis of all MDR A. baumannii isolates recovered during an outbreak from 2011 to 2015 in a tertiary care cancer hospital. Cases were classified as colonized or infected. We determined sequence types following the Bartual scheme and plasmid profiles. RESULTS: There were 106 strains of A. baumannii isolated during the study period. Sixty-six (62.3%) were considered as infection and 40 (37.7%) as colonization. The index case, identified by molecular epidemiology, was a patient with a drain transferred from a hospital outside Mexico City. Ninety-eight additional cases had the same MultiLocus Sequence Typing (MLST) 758, of which 94 also had the same plasmid profile, two had an extra plasmid, and two had a different plasmid. The remaining seven isolates belonged to different MLSTs. Fifty-three patients (50%) died within 30 days of A. baumanniii isolation: 28 (20%) in colonized and 45 (68.2%) in those classified as infection (p<0.001). In multivariate regression analysis, clinical infection and patients with hematologic neoplasm, predicted 30-day mortality. The molecular epidemiology of this outbreak showed the threat posed by the introduction of MDR strains from other institutions in a hospital of immunosuppressed patients and highlights the importance of adhering to preventive measures, including contact isolation, when admitting patients with draining wounds who have been hospitalized in other institutions.


Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/mortalidade , Infecção Hospitalar/epidemiologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Acinetobacter baumannii/patogenicidade , Adulto , Idoso , Estudos de Casos e Controles , Surtos de Doenças , Resistência a Múltiplos Medicamentos/fisiologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Hospitais Gerais , Humanos , Masculino , México , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Epidemiologia Molecular/métodos , Tipagem de Sequências Multilocus/métodos , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Estudos Retrospectivos , Análise de Sequência de DNA/métodos , beta-Lactamases/genética
13.
J Environ Public Health ; 2020: 2571293, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32612664

RESUMO

Waterborne Escherichia coli are a major reservoir of antimicrobial resistance (AMR). Carbapenem-resistance, especially when mediated by transferable carbapenemase-encoding genes, is spreading worldwide and causing dramatically limiting treatment options. In our country, studies for the detection of carbapenem resistance in drinking water do not exist; therefore, this work was carried out to determine the prevalence of carbapenem-resistant genes "blaKPC, blaIMP, blaNDM, blaSPM, blaVIM, and blaOXA-48" among Escherichia coli isolated from drinking water in Khartoum, Sudan. A total of forty-five E. coli bacteria were isolated from different sources of drinking water. Antimicrobial susceptibility testing was performed using imipenem (10 mg/disc), gentamicin (10 mg/disc), ceftriaxone (30 mg/disc), ciprofloxacin (5 mg/disc), chloramphenicol (30 mg/disc), and tetracycline (30 mg/disc). "Sensitive" or "resistant" patterns of E. coli were judged using antibiotic minimum inhibitory concentration (MIC). Bacterial genomic DNA was extracted by the boiling method, and then multiplex polymerase chain reaction was performed to detect the carbapenemase genes (blaKPC, blaIMP, bla NDM , blaSPM, blaVIM, and blaOXA-48). Multiplex PCR assays confirmed the presence of carbapenemase genes in 28% of all water isolates. OXA-48 gene was the most predominant gene, detected in 15.5% of the isolates. The blaKPC and bla SPM genes were also detected in 4.4% and 8.8% of the isolates, respectively. However, the isolates were negative for bla NDM , blaVIM, and blaIMP genes. The isolates showed a high rate of tetracycline resistance (97.7%), followed by gentamicin (57.7%), ciprofloxacin (46.6%), ceftriaxone (35.5%), and chloramphenicol (31.1%). In conclusion, this study confirmed for the first time the presence of E. coli carried carbapenem-resistant genes in the drinking water of Khartoum state, Sudan. These isolates commonly carried OXA-48 (7/45), followed by SPM (4/45) and KPC (2/45).


Assuntos
Carbapenêmicos/farmacologia , Água Potável/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Escherichia coli/genética , Humanos , Testes de Sensibilidade Microbiana , Sudão , beta-Lactamases/genética
14.
Ecotoxicol Environ Saf ; 201: 110817, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32512417

RESUMO

Cellular exposure to xenobiotic human-made products will lead to oxidative stress that gives rise to DNA damage, as well as chemical or mechanical damage. Distinguishing the chemicals that will induce oxidative stress and predicting their toxicity is necessary. In the present study, 4270 compounds in the ARE-bla assay were investigated to predict active and inactive compounds by using simple algorithms, namely, recursive partitioning (RP) and binomial logistic regression, and to develop the quantitative structure-activity relationship (QSAR) models of chemicals that activate the ARE pathway to induce oxidative stress and exert toxic effects on cells. A decision tree based on scaffold-based fragments obtained through RP analysis showed the best identification accuracy. However, the overall identification accuracy of this model for active compounds was unsatisfactory due to limited fragments. Furthermore, a binomial logistic regression model was developed from 638 active compounds and 3632 inactive chemicals. The model with a cutoff of 0.15 could predict chemicals that were active or inactive with the prediction accuracy of 69.1%. Its area under the receiver operating characteristic (ROC) curve metric (AUROC) was 0.762, which indicated the acceptable predictive ability of this model. The parameters nBM (number of multiple bonds) and H% (percentage of H atom) played dominant roles in the prediction of the activity (inactive or active) of chemicals. A global QSAR model was developed to predict the toxicity of active chemicals. However, the model displayed an unsatisfactory result with R2 = 0.316 and R2ext = 0.090. Active chemicals were then classified on the basis of structure. A total of 79 compounds with carbon chains could be predicted with acceptable performance by using a QSAR model with six descriptors (R2 = 0.722, R2ext = 0.798, Q2Loo = 0.654, Q2Boot = 0.755, Q2ext = 0.721). The simple models established here contribute to efforts on identification compounds inducing oxidative stress and provide the scientific basis for risk assessment to organisms in the environment.


Assuntos
Compostos Orgânicos , Estresse Oxidativo/efeitos dos fármacos , Algoritmos , Elementos de Resposta Antioxidante/genética , Bioensaio , Bases de Dados Factuais , Genes Reporter , Humanos , Modelos Logísticos , Compostos Orgânicos/química , Compostos Orgânicos/toxicidade , Estresse Oxidativo/genética , Relação Quantitativa Estrutura-Atividade , beta-Lactamases/genética
15.
Rev Soc Bras Med Trop ; 53: e20190526, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32578705

RESUMO

INTRODUCTION: This study investigated the genetic environment of bla KPC-2 in Klebsiella pnemoniae multi-drug resistant clinical isolates. METHODS: Four carbapenemase gene isolates resistant to carbapenems, collected from infected patients from two hospitals in Brazil, were investigated using polymerase chain reaction and plasmid DNA sequencing. RESULTS: The bla KPC-2 gene was located between ISKpn6 and a resolvase tnpR in the non-Tn4401 element (NTEKPC-IId). It was detected on a plasmid belonging to the IncQ1 group. CONCLUSIONS: To our knowledge, this is the first report of the presence of the bla KPC-2 gene in the NTEKPC-IId element carried by plasmid IncQ1 from infections in Brazil.


Assuntos
Antibacterianos/farmacologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/genética , beta-Lactamases/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Humanos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Reação em Cadeia da Polimerase
16.
Water Res ; 182: 115827, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32580076

RESUMO

A growing body of evidence has demonstrated that extraintestinal pathogenic E. coli (ExPEC), such as the urinary pathogenic E. coli (UPEC), are common constituents of treated wastewater, and therefore represent a potential public health risk. However, no single virulence gene, or set of virulence genes, can be used to conclusively identify this genetically diverse pathotype. As such we sought to identify and characterize the public health relevance of potential UPEC found in treated sewage/wastewater using a comparative genomics approach. Presumptive wastewater UPEC (W-UPEC) were initially identified by virulence gene screening against 5 virulence genes, and for which isolates containing ≥3 virulence genes were whole genome sequenced (n = 24). Single nucleotide polymorphic (SNP) spanning tree analysis demonstrated that many of these wastewater UPEC (WUPEC) were virtually identical at the core genome (0.4 Mbp) when compared to clinical UPEC (C-UPEC) sequences obtained from NCBI, varying by as little as 1 SNP. Remarkably, at the whole genome level, W-UPEC isolates displayed >96% whole genome similarity to C-UPEC counterparts in NCBI, with one strain demonstrating 99.5% genome similarity to a particular C-UPEC strain. The W-UPEC populations were represented by sequence types (ST) known to be clinically important, including ST131, ST95, ST127 and ST640. Many of the W-UPEC carried the exact same complement of virulence genes as their most closely related C-UPEC strains. For example, O25b-ST131 W-UPEC strains possessed the same 80 virulence genes as their most closely related C-UPEC counterparts. Concerningly, W-UPEC strains also carried a plethora of antibiotic resistance genes, and O25b-ST131strains were designated as extended spectrum beta-lactamase (ESBL) producing E. coli by both genome profiling and phenotypic resistance testing. W-UPEC ST131 strains were found in the effluents of a single treatment plant at different times, as well as different wastewater treatment plants, suggesting a differentially ability to survive wastewater treatment. Indeed, in sewage samples treated with chlorine doses sufficient for inducing a ∼99.99% reduction in total E. coli levels, UPEC represented a significant proportion of the chlorine-resistant population. By contrast, no Shiga toxin-producing E. coli were observed in these chlorinated sewage libraries. Our results suggest that clinically-relevant UPEC exist in treated wastewater effluents and that they appear to be specifically adapted to survive wastewater treatment processes.


Assuntos
Infecções por Escherichia coli , Purificação da Água , Escherichia coli , Genótipo , Humanos , Fatores de Virulência , Águas Residuárias , beta-Lactamases/genética
17.
Proc Natl Acad Sci U S A ; 117(21): 11597-11607, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385156

RESUMO

The distribution of fitness effects of mutation plays a central role in constraining protein evolution. The underlying mechanisms by which mutations lead to fitness effects are typically attributed to changes in protein specific activity or abundance. Here, we reveal the importance of a mutation's collateral fitness effects, which we define as effects that do not derive from changes in the protein's ability to perform its physiological function. We comprehensively measured the collateral fitness effects of missense mutations in the Escherichia coli TEM-1 ß-lactamase antibiotic resistance gene using growth competition experiments in the absence of antibiotic. At least 42% of missense mutations in TEM-1 were deleterious, indicating that for some proteins collateral fitness effects occur as frequently as effects on protein activity and abundance. Deleterious mutations caused improper posttranslational processing, incorrect disulfide-bond formation, protein aggregation, changes in gene expression, and pleiotropic effects on cell phenotype. Deleterious collateral fitness effects occurred more frequently in TEM-1 than deleterious effects on antibiotic resistance in environments with low concentrations of the antibiotic. The surprising prevalence of deleterious collateral fitness effects suggests they may play a role in constraining protein evolution, particularly for highly expressed proteins, for proteins under intermittent selection for their physiological function, and for proteins whose contribution to fitness is buffered against deleterious effects on protein activity and protein abundance.


Assuntos
Evolução Molecular , Aptidão Genética/genética , Mutação de Sentido Incorreto/genética , Mutação de Sentido Incorreto/fisiologia , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismo
18.
PLoS One ; 15(5): e0232710, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32384111

RESUMO

With the growing threat of antimicrobial resistance worldwide, uncovering the molecular epidemiology is critical for understanding what is driving this crisis. We aimed to evaluate the prevalence of plasmid-mediated-quinolone-resistance (PMQR) and extended-spectrum beta-lactamase- (ESBL) producing gram-negative organisms among primigravid women with bacteriuria. We collected urine specimens from primigravid women attending their first antenatal visit at Gandhi Hospital during October 1, 2015 to September 30, 2016. We determined antimicrobial susceptibility and ESBL and quinolone resistance using VITEK-2. We performed polymerase chain reaction amplification on resistant isolates for detection of ESBL-encoding genes (TEM, SHV, CTX-M) and PMQR genes (qnrA, qnrB, qnrD, qnrS, aac (6')-Ib-cr). Of 1,841 urine samples, 133 demonstrated significant bacterial growth with gram-negative bacilli accounting for 85% of isolates, including Escherichia coli (n = 79), Klebsiella pneumoniae (n = 29), Sphingomonas (n = 3), Enterobacter (n = 1), and Citrobacter (n = 1). We found 65% of E. coli isolates and 41% of K. pneumoniae isolates were ESBL positive. Of ESBL-positive isolates, the most common genes conferring resistance were TEM-1 (66.7%) followed by CTX-M-15 (33.3%). Fifty-seven percent of ESBL-positive E. coli also demonstrated resistance to quinolones with the most common PMQR genes being qnr-S (62.5%) and aac (6')-Ib-cr (37.5%). We did not find any resistance to quinolones among ESBL-positive K. pneumoniae isolates. Across different classes of antibiotics we found a strong clustering of multi-drug resistance in E. coli with over 45% of ESBL-positive isolates demonstrating resistance to at least three classes of antibiotics. This study emphasizes the high prevalence of plasmid-mediated ESBL and quinolone resistance in community-acquired urinary tract infections of primigravid women. The overall abundance of multi-drug-resistant isolates in this population is alarming and may present therapeutic challenges.


Assuntos
Resistência Microbiana a Medicamentos/genética , Plasmídeos/genética , Infecções Urinárias/microbiologia , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Feminino , Genes Bacterianos , Humanos , Índia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Fenótipo , Gravidez , Quinolonas/farmacologia , beta-Lactamases/genética
19.
J Med Microbiol ; 69(6): 792-796, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32459618

RESUMO

Introduction. Klebsiella rods, belonging to the family Enterobacteriaceae, are generally opportunistic pathogens commonly associated with nosocomial infections, especially in intensive care units. Interestingly, strains of this genus also show multi-drug resistance. In recent years, multiple studies have indicated that the prevalence of carbapenem resistance has increased rapidly among Klebsiella representatives.Aim. The aim of this study was to assess the usefulness of selected phenotypic and genotypic methods for the detection of the most important carbapenemases in Klebsiella strains.Methodology. The study involved 51 Klebsiella strains. The ability to produce carbapenemases was determined by phenotypic methods (double disc synergy test, test with four discs and three inhibitors, CarbaNP test, culture on chromogenic medium, panels of automatic method - Phoenix, CIM test and modified Hodge test). The potential for carbapenemase synthesis was also evaluated using real-time PCR, detecting bla VIM/IMP, bla KPC, bla NDM and bla OXA-48 genes.Results. Using the phenotypic methods, positive results were obtained for all of the analysed strains. Using PCR, carbapenemase synthesis potential was confirmed on the molecular level; the bla VIM gene was detected in 23 strains, the bla NDM gene in 26 strains and the bla OXA-48 gene in two strains.Conclusion. There was complete agreement between the carbapenemases detected by the genetic method and the results obtained with phenotypic methods.


Assuntos
Proteínas de Bactérias/análise , Klebsiella/enzimologia , beta-Lactamases/análise , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Genótipo , Fenótipo , beta-Lactamases/biossíntese , beta-Lactamases/genética
20.
PLoS One ; 15(5): e0231852, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469885

RESUMO

BACKGROUND: The crisis of antimicrobial resistance is already here with us, affecting both humans and animals alike and very soon, small cuts and surgeries will become life threatening. This study aimed at determine the whole genome sequences of multi-drug resistant Escherichia coli isolated in a Pastoralist Community of Western Uganda: phylogenomic changes, virulence and resistant genes. METHODS: This was a laboratory based cross sectional study. Bacterial isolates analyzed in this study were 42 multidrug resistant E. coli isolated from stool samples from both humans (n = 30) and cattle (n = 12) in pastoralist communities collected between January 2018-March 2019. Most of the isolates (41/42) were resistant to three or more antibiotics (multi-drug resistant) and 21/42 isolates were ESBL producers; 13/30 from human and 8/12 from cattle. Whole Genome Sequencing (WGS) was carried out at the facilities of Kenya Medical Research Institute-Wellcome trust, Kilifi, to determine the phylogenomic changes, virulence and resistant genes. RESULTS: At household level, the genomes from both human and animals clustered away from one another except for one instance where two human isolates from the same household clustered together. However, 67% of the E. coli isolated from cattle were closely related to those found in humans. The E. coli isolates were assigned to eight different phylogroups: A, B1, B2, Cladel, D, E, F and G, with a majority being assigned to phylogroup A; while most of the animal isolates were assigned to phylogroup B1. The carriage of multiple AMR genes was higher from the E. coli population from humans than those from cattle. Among these were Beta-lactamase; blaOXA-1: Class D beta-lactamases; blaTEM-1, blaTEM-235: Beta-lactamase; catA1: chloramphenicol acetyl transferase; cmlA1: chloramphenicol efflux transporter; dfrA1, dfrA12, dfrA14, dfrA15, dfrA17, dfrA5, dfrA7, dfrA8: macrolide phosphotransferase; oqxB11: RND efflux pump conferring resistance to fluoroquinolone; qacL, qacEdelta1: quinolone efflux pump; qnrS1: quinolone resistance gene; sul1, sul2, sul3: sulfonamide resistant; tet(A), tet(B): tetracycline efflux pump. A high variation of virulence genes was registered among the E. coli genomes from humans than those of cattle origin. CONCLUSION: From the analysis of the core genome and phenotypic resistance, this study has demonstrated that the E. coli of human origin and those of cattle origin may have a common ancestry. Limited sharing of virulence genes presents a challenge to the notion that AMR in humans is as a result of antibiotic use in the farm and distorts the picture of the directionality of transmission of AMR at a human-animal interface and presents a task of exploring alternative routes of transmission of AMR.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli , Filogenia , Fatores de Virulência/genética , Animais , Antibacterianos/farmacologia , Bovinos , Estudos Transversais , Escherichia coli/genética , Escherichia coli/patogenicidade , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/transmissão , Proteínas de Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano , Humanos , Testes de Sensibilidade Microbiana , Uganda , Sequenciamento Completo do Genoma , beta-Lactamases/genética
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