Azidothymidine produces synergistic activity in combination with colistin against antibiotic-resistant Enterobacteriaceae.

Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, antibiotic-resistant Enterobacteriaceae is prevalent and extremely difficult to treat. Reusing existing drugs and rejuvenating the therapeutic potential of existing antibiotics represent an attractive novel strategy. Azidothymidine (AZT) is an antiretroviral drug which is used in combination with other antivirals to prevent and to treat HIV/AIDS. AZT is also active against Gram-negative bacteria but has not been developed for that purpose. Here we investigated in vitro and in vivo efficacy of AZT in combination with colistin against antibiotic-resistant Enterobacteriaceae including extended-spectrum beta-lactamase (ESBL), New Delhi metallo-beta-lactamase 1 (NDM) or the mobilized colistin resistance (mcr-1) producing strains. Minimum inhibitory concentration was determined using the broth microdilution method. The combinatory effect of AZT and colistin was examined using the checkerboard method and time-kill analysis. A murine peritoneal infection model was used to test the therapeutic effect of the combination of AZT and colistin. Fractional inhibitory concentration index from checkerboard assay demonstrated that AZT synergized with colistin against 61% and 87% of ESBL-producing Escherichia coli and Klebsiella pneumoniae, respectively, 100% of NDM-1-producing strains and 92% of mcr-1 producing E. coli Time-kill analysis demonstrated significant synergistic activities when AZT was combined with colistin. In the murine peritoneal infection model, AZT in combination with colistin showed augmented activities of both drugs in the treatment of NDM-1 K. pneumoniae and mcr-1 E. coli infections. AZT and colistin combination poses a potential to be used coherently to treat antibiotic-resistant Enterobacteriaceae infections

Plasmídeos IncP-1 em Enterobacteriaceae resistentes a antibióticos

Antibiotic resistance is increasing worldwide closely linked with antibiotic misuse and overuse. Antibiotic gene resistance is commonly disseminated through MGEs (mobile genetic elements), especially plasmids. IncP-1 plasmids are BHR presumably found in several bacterial families and have been associated with antibiotic resistance and tolerance to metals. The identification of plasmid groups has been frequently done using the PBRT (PCR-based replicon typing) approach which assigns plasmids to Inc groups, including the IncP-1 plasmid group. The efforts to further characterize these structures, especially when associated with antibiotic resistant bacteria, has been lacking, and consequently, information is scarce and dispersed. Therefore, a systematic analysis was carried out to understand the occurrence, distribution, and genetic traits of IncP-1 plasmids associated with antibiotic resistant bacteria. To do so, a bibliographic search strategy was followed, where the Scopus platform was used to look for studies that used the PBRT method developed by Carattoli et al. (2005) for plasmid identification and that actually detected IncP-1 plasmids structures. Article collection for a period of 5 years resulted in 96 eligible articles, which were used to retrieve relevant information about IncP-1 plasmids occurrence and features. The articles combined reported the identification of 629 IncP-1 replicons. Altogether, the bacterial hosts of IncP-1 plasmids were found in 32 countries and were collected from a variety of environmental sources. Bacterial hosts belonged to 28 species distributed in 10 genera, of Enterobacteriaceae and Pseudomonadaceae families and were resistant to 10 different antibiotic classes, harboring 32 different resistance genes. IncP-1 plasmid-positive bacteria usually harbored at least 2 different Inc groups. Of all the IncP-1 plasmids identified, 229 (~36%) were further described, their sizes ranging from 35 to 320 kb and have been associated with medically important resistance genes and additional genetic elements that could potentiate gene dissemination. Furthermore, we studied the molecular diversity of previously reported IncP-1 plasmids occurring in 9 Escherichia coli isolates from an UWTP in Portugal. This was accomplished via PCR amplification of the 281 bp trfA gene fragment sequences and transfer to new well-known bacterial hosts for further characterizations. Amplicon sequencing showed 100% identity in all trfA fragments suggesting genetic structure conservation association to similar bacteria and environments. Similarity searching of the trfA fragment sequence was used to select closely related fully sequenced IncP-1β1 plasmids for comparisons. As a result, 63 closely related replicons were selected for comparative analysis and found to be usually large genetic structures, isolated mainly from wastewater and soil, harboring genetic determinants associated with resistance to aminoglycosides, sulphonamides, and β-lactams, and with tolerance to Hg. Attempts to transfer the 9 IncP-1 plasmids to a recipient strain were unsuccessful probably due to the chosen selective markers. The high prevalence of mer operon genes in these IncP-1β1 plasmids, suggesting mercury tolerance, could be used in a future work as a selective marker to transfer these 9 IncP-1 plasmids another bacterial host allowing for proper genomic characterization of these promiscuous structures.A resistência aos antibióticos está a aumentar em todo o mundo, intimamente associada ao uso incorreto e excessivo de antibióticos. Os genes de resistência a antibióticos são comummente disseminados por meio de EGMs (elementos genéticos móveis), especialmente plasmídeos. Os plasmídeos IncP-1 têm amplo espetro de hospedeiros, são presumivelmente encontrados em várias famílias de bactérias, e têm sido associados à resistência a antibióticos e tolerância a metais. A identificação de grupos de plasmídeos tem sido frequentemente realizada utilizando a abordagem PBRT (PCR-based replicon typing), que atribui plasmídeos a grupos de incompatibilidade, incluindo o grupo IncP-1. Os esforços para caracterizar melhor essas estruturas, principalmente quando associadas a bactérias resistentes a antibióticos, têm falhado e, consequentemente, as informações são escassas e dispersas. Portanto, uma análise sistemática foi realizada para compreender a ocorrência, distribuição e características genéticas de plasmídeos IncP-1 associados a bactérias resistentes a antibióticos. Para tal, a plataforma Scopus foi utilizada para realizar uma pesquisa bibliográfica, para selecionar estudos que identificaram plasmídeos IncP-1 através do método PBRT desenvolvido por Carattoli et al. (2005). A pesquisa abrangeu um período de 5 anos e resultou em 96 artigos elegíveis, os quais foram usados para a obtenção de informações relevantes sobre a ocorrência e características dos plasmídeos IncP-1. Os artigos combinados relataram a identificação de 629 replicões IncP-1. Ao todo, os hospedeiros bacterianos dos plasmídeos IncP-1 foram encontrados em 32 países e foram coletados de uma variedade de fontes ambientais. Os estudos identificaram hospedeiros bacterianos pertencentes a 28 espécies, distribuídas em 10 géneros das famílias Enterobacteriaceae e Pseudomonadaceae, foram identificados como resistentes a 10 classes de antibióticos, possuindo 32 genes de resistência diferentes, e pelo menos apresentando replicões de 2 grupos Inc diferentes. De todos os plasmídeos IncP-1 identificados, 229 (~ 36%) foram descritos posteriormente, e seus tamanhos variam de 35 a 320 kb, foram associados a genes de resistência clinicamente importantes e a elementos genéticos adicionais que poderiam potenciar a disseminação de genes. Além disso, foi estudada a diversidade molecular de plasmídeos IncP-1 identificados anteriormente em 9 isolados de Escherichia coli de uma UWTP em Portugal. Isso foi realizado por meio da amplificação por PCR de um fragmento do gene trfA de 281 bp, e tentativa de transferência para novos hospedeiros bacterianos bem conhecidos para posterior caracterização. A sequenciação dos amplicões mostrou 100% identidade entre fragmentos trfA sugerindo conservação das suas estruturas genéticas e associação com bactérias e ambientes semelhantes. A pesquisa por similaridade da sequência do fragmento trfA foi usada para selecionar e comparar plasmídeos IncP-1β1 totalmente sequenciados. Isso resultou na análise de 63 replicões, geralmente grandes estruturas genéticas, isolados de águas residuais e solos, possuindo determinantes genéticos associados à resistência a aminoglicosídeos, sulfonamidas e βlactâmicos, e à tolerância ao Hg. As tentativas de transferir os 9 plasmídeos IncP-1 para uma estirpe de laboratório não tiveram sucesso, provavelmente devido aos marcadores seletivos escolhidos. A ampla prevalência de genes do operão mer nestes plasmídeos IncP-1β1, sugerindo tolerância ao mercúrio, poderia ser usada em um trabalho futuro como um marcador seletivo para a transferência dos 9 plasmídeos IncP-1 de água residual para outro hospedeiro, permitindo a caracterização genética dessas estruturas.Mestrado em Microbiologi

Evaluation of drug-resistant Enterobacteriaceae in retail poultry and beef

There has been increasing concern on the emergence of multidrug-resistant foodborne pathogens from foods of animal origin, including poultry. The current study aimed to evaluate antibiotic-resistant Enterobacteriaceae from raw retail chicken/turkey parts (thigh, wings, breast, and ground) and beef meat (ground and chunks) in Middle Tennessee. Resistance of the collected Enterobacteriaceae to a panel of antibiotics was determined by the Kirby-Bauer disk diffusion test. Retail meats were also assayed for the presence of Salmonella spp. and Escherichia coli O157:H7. Two hundred thirty-seven samples representing 95.2% of the total of 249 samples tested were positive for Enterobacteriaceae. The level of contamination with Enterobacteriaceae in raw meats ranged from 3.26 log10 cfu/g to 4.94 log10 cfu/g with significant differences in counts among meat types (P \u3c 0.05). Contamination was significantly greater (P \u3c 0.05) in ground beef, beef chucks, ground chicken, chicken breast, and turkey wings (4.92, 4.58, 4.94, 4.75, 4.13 log10 cfu/g, respectively) than ground turkey and chicken wings (3.26 and 3.26 log10 cfu/g, respectively). Klebsiella oxytoca, Serratia spp., E. coli, and Haffnia alvei were most prevalent contaminants at 27.4, 14.3, 12.1, and 11.4%, respectively. Resistance of the Enterobacteriaceae to antimicrobials was most frequent with erythromycin, penicillin, and ampicillin at 100, 89, and 65.8%, respectively. Few (2.7%) of the Enterobacteriaceae were resistant to chloramphenicol. Salmonella spp., E. coli O157:H7, Morganella morganii, Yersinia enterocolitica, and Vibrio parahemolyticus exhibited multiple drug resistance. This investigation demonstrates that raw poultry and beef are potential reservoirs of antibiotic-resistant Enterobacteriaceae

In vitro susceptibility of recent antibiotic-resistant urinary pathogens to ertapenem and 12 other antibiotics

7 p.Background: The treatment of complicated urinary tract infections may require the use of a parenteral anti-biotic with potent activity against the most common urinary pathogens. Ertapenem is a broad-spectrum 1?-methyl carbapenem with a long plasma half-life that allows administration of a single daily dose. Methods: The purpose of this work was to test the in vitro susceptibility to ertapenem, ampicillin, cefazolin, cefuroxime, cefotaxime, co-amoxiclav, piperacillin/tazobactam, imipenem, gentamicin, amikacin, fosfo-mycin, ciprofloxacin and co-trimoxazole of 482 strains of urinary pathogens of the family Enterobacteriaceae isolated from patients in the community of Madrid (40% from males). The distribution was as follows: Escherichia coli (n = 315), Proteus mirabilis (n = 42), Klebsiella spp. (n = 14) and AmpC-producing Entero-bacteriaceae (n = 111). The strains studied were selected based on their resistance to quinolones and aminoglycosides, and their production of extended-spectrum ?-lactamases (ESBLs) or AmpC-type ?-lacta-mases. Results: All the strains were susceptible to ertapenem, imipenem and amikacin. The MIC90 of ertapenem ranged from a minimum of 0.03 mg/L for Proteus vulgaris and a maximum of 1 mg/L for Enterobacter spp. Ertapenem was the most active of all drugs tested in all cases. On comparing antibiotic resistance among ESBL-producing strains of E. coli (n = 35) and E. coli strains not producing ESBLs (n = 280), statistically significant differences were obtained for ciprofloxacin (P = 0.002) and gentamicin (P = 0.011). Regarding ertapenem, only a slight increase in MIC50 was seen, the value being 0.015 mg/L for strains not producing ESBLs versus 0.03 mg/L for ESBL-producing strains. Conclusions: In view of its significant antibiotic potency against antibiotic-resistant Enterobacteriaceae, ertapenem may constitute a good therapeutic alternative in urinary infections caused by these pathogens

The effect of bambermycin, carbadox, chlortetracycline and olaquindox on antibiotic resistance in intestinal coliforms: a new animal model

Groups of germ-free mice kept in isolators and associated with faecal microflora from piglets were continuously given either water or a solution of one of the following: chlortetracycline (20 micrograms/ml), carbadox (50 micrograms/ml), olaquindox (50 micrograms/ml), bambermycin (flavomycin) (5 micrograms/ml) or mixtures of these drugs. The proportions of lactose-fermenting bacteria in their faeces which were resistant to chlortetracycline, carbadox or olaquindox were measured by a comparative plate-counting procedure. Compared to occurrence in control mice, the occurrence of antimicrobial drug-resistant bacteria was higher in mice receiving chlortetracycline (P less than 0.001) and lower in mice receiving bambermycins (P less than 0.005). In contrast, olaquindox and carbadox did not change the proportion of resistant coliforms in mice faeces. A control experiment was conducted with five groups of germ-free mice given the same flora and kept without drugs in separate isolators. No difference in the occurrence of resistant coliforms could be found between these groups. The germ-free mouse associated with faecal microflora from a conventional animal seems to be a suitable model for determining in vivo the effect of low doses of antimicrobial drugs on drug resistance in lactose-fermenting enteric flora

Prevalence and risk factors for intestinal carriage of CTX-M-type ESBLs in Enterobacteriaceae from a Thai community

The incidence of infections caused by antimicrobial-resistant Enterobacteriaceae in Thailand is increasing and human intestinal flora is an important reservoir for these organisms. This study was carried out to determine the intestinal carriage of bla CTX-M extended spectrum ß-lactamase-positive Enterobacteriaceae (ESBL + E) and AmpC-positive Enterobacteriaceae in a community setting in Northern Thailand, and to identify potential risk factors for carriage. A total of 307 fecal samples were collected from healthy volunteers in Phitsanulok province, and cefotaxime-resistant Enterobacteriaceae (CtxRE) were isolated using selective media. Polymerase chain reaction (PCR) was used to detect ESBL and AmpC genes. Risk factors were analyzed using multiple logistic regression. Genotyping was performed by multilocus sequence typing (MLST) analysis. Two hundred ninety-one CtxRE isolates were obtained and Escherichia coli was the predominant organism (66.3%). The intestinal carriage rates of bla CTX-M ESBL + E and AmpC-positive Enterobacteriaceae were 52.1% and 6.2%, respectively. Comparative levels of bla CTX-M group 1 and bla CTX-M group 9 were found while bla CMY-2 was the predominant genotype among AmpC genes. Co-existence of two ß-lactamase genes in a single isolate was found in 6.5% of isolates. Consumption of undercooked meat was strongly associated with intestinal carriage of bla CTX-M ESBL + E (p = 0.003, OR = 2.133, 95% CI = 1.289–3.530). Phylogenetic grouping and MLST analysis of E. coli isolates revealed the presence of E. coli B2-ST131 (n = 8). Of these, seven carried bla CTX-M-group 9 and 1 carried bla CMY-2. Our results suggest that residents in Thailand are at high risk for developing endogenous infections caused by antibiotic-resistant Enterobacteriaceae

Role of the gut microbiota in nutrient competition and protection against intestinal pathogen colonization.

The human gut microbiota can restrict the growth of pathogens to prevent them from colonizing the intestine ('colonization resistance'). However, antibiotic treatment can kill members of the gut microbiota ('gut commensals') and reduce competition for nutrients, making these nutrients available to support the growth of pathogens. This disturbance can lead to the growth and expansion of pathogens within the intestine (including antibiotic-resistant pathogens), where these pathogens can exploit the absence of competitors and the nutrient-enriched gut environment. In this review, we discuss nutrient competition between the gut microbiota and pathogens. We also provide an overview of how nutrient competition can be harnessed to support the design of next-generation microbiome therapeutics to restrict the growth of pathogens and prevent the development of invasive infections