Plasmid mediated antibiotic resistance : with focus on extended spectrum β-lactamases (ESBL)

Enterobacteriaceae is a family of bacteria including Escherichia coli and Klebsiella pneumoniae that are common colonizers of the gastrointestinal tract. Extended spectrum β- lactamases (ESBL) are bacterial enzymes that degrade β-lactam antibiotics and thus make the bacteria resistant to β-lactam antibiotics. ESBL-producing Enterobacteriaceae (EPE) is an increasing problem constituting a burden on health care systems conferring excess morbidity, prolonged hospital stay and mortality. There are many hundreds of different ESBL-genes described. The most common are the blaCTX-M group. ESBL-genes are often situated on plasmids. This enables a fast dissemination since they can be spread both vertically and horizontally. Horizontal dissemination also enables them to spread between bacteria of different species. Outbreaks of resistant bacteria, such as EPE, must be rapidly detected and contained. Today the main focus during an outbreak is to conduct epidemiological typing of the bacterial strain. However, a horizontal outbreak of resistance genes in the bacterial population can also occur and go undetected if only the strain is subjected to epidemiological typing. There is a need for better understanding of plasmid dissemination and their role in transferring antibiotic resistance genes. In this thesis I have evaluated different methods for epidemiological typing of both strains and plasmids by analyzing different nationwide collections of Swedish EPEs from 2007 to 2011. In Sweden EPE became notifiable according to the communicable disease act in 2007. The number of reported cases has since increased from ~2000 to ~7000 cases per year. In paper I and II we found that both bacterial strain types and ESBL-genotypes were stable over the five- year period. However, a decline in median age for contracting an EPE infection in the Swedish population was seen. This suggests that EPE-carriage is increasing among healthy people in the community. The decline of EPE amongst elderly people might also be an effect of increased awareness and improved infection control in hospitals limiting outbreaks amongst elderly and multi-ill individuals. Paper III describes plasmids of EPE in detail and presents a new plasmid typing approach using next generation sequencing (NGS). The Swedish EPE were found to often carry several plasmids as well as multi-replicon plasmids of IncF-type. Small cryptic plasmids were common in the EPE isolates which raises the question of their role in the bacterial cell. Paper IV describes the strain-plasmid-interplay in the gut flora of three EPE carriers over time. The three patients presented different examples of this interplay. In one of the patients isolates with identical epidemiological strain types were found to also have identical plasmids. In a second patient plasmid variation was observed between identical epidemiological strain types. A third patient had a mixed EPE flora with different epidemiological strain types and different species where similar plasmids were seen in divergent strains as well as the opposite. These results most likely reflect the effect of antibiotic usage and EPE risk factors, such as travel abroad, on the gut flora in terms of bacterial communication and dissemination of ESBL-genes. This thesis has contributed to increased knowledge of the Swedish epidemiology of EPE and increased awareness of the impact of plasmids in the dissemination of common resistance genes

Использование нетрадиционных методов лечения в дерматовенерологии

ВЕНЕРОЛОГИЯДЕРМАТОЛОГИЯМЕДИЦИНА ТРАДИЦИОННА

Worsening epidemiological situation of carbapenemase-producing Enterobacteriaceae in Europe, assessment by national experts from 37 countries, July 2018

European Antimicrobial Resistance Genes Surveillance Network (EURGen-Net) capacity survey group (Portugal): Manuela Caniça, Vera Manageiro,A survey on the epidemiological situation, surveillance and containment activities for carbapenemase-producing Enterobacteriaceae (CPE) was conducted in European countries in 2018. All 37 participating countries reported CPE cases. Since 2015, the epidemiological stage of CPE expansion has increased in 11 countries. Reference laboratory capability, dedicated surveillance and a specific national containment plan are in existence in 33, 27 and 14 countries, respectively. Enhanced control efforts are needed for CPE containment in Europe.info:eu-repo/semantics/publishedVersio

Molecular Characterisation of Trimethoprim Resistance in Escherichia coli and Klebsiella pneumoniae during a Two Year Intervention on Trimethoprim Use

BACKGROUND: Trimethoprim resistance is increasing in Enterobacteriaceae. In 2004-2006 an intervention on trimethoprim use was conducted in Kronoberg County, Sweden, resulting in 85% reduction in trimethoprim prescriptions. We investigated the distribution of dihydrofolate reductase (dfr)-genes and integrons in Escherichia coli and Klebsiella pneumoniae and the effect of the intervention on this distribution. METHODOLOGY/PRINCIPAL FINDINGS: Consecutively isolated E. coli (n = 320) and K. pneumoniae (n = 54) isolates phenotypically resistant to trimethoprim were studied. All were investigated for the presence of dfrA1, dfrA5, dfrA7, dfrA8, dfrA12, dfrA14, dfrA17 and integrons class I and II. Isolates negative for the seven dfr-genes (n = 12) were also screened for dfr2d, dfrA3, dfrA9, dfrA10, dfrA24 and dfrA26. These genes accounted for 96% of trimethoprim resistance in E. coli and 69% in K. pneumoniae. The most prevalent was dfrA1 in both species. This was followed by dfrA17 in E. coli which was only found in one K. pneumoniae isolate. Class I and II Integrons were more common in E. coli (85%) than in K. pneumoniae (57%). The distribution of dfr-genes did not change during the course of the 2-year intervention. CONCLUSIONS/SIGNIFICANCE: The differences observed between the studied species in terms of dfr-gene and integron prevalence indicated a low rate of dfr-gene transfer between these two species and highlighted the possible role of narrow host range plasmids in the spread of trimethoprim resistance. The stability of dfr-genes, despite large changes in the selective pressure, indirectly suggests a low fitness cost of dfr-gene carriage

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Infections caused by Extended spectrum beta-lactamase (ESBL)-producing E. coli are an emerging global problem, threatening the effectiveness of the extensively used beta-lactam antibiotics. ESBL dissemination is facilitated by plasmids, transposons, and other mobile elements. We have characterized the plasmid content of ESBL-producing E. coli from human urinary tract infections. Ten diverse isolates were selected; they had unrelated pulsed-field gel electrophoresis (PFGE) types (<90% similarity), were from geographically dispersed locations and had diverging antibiotic resistance profiles. Three isolates belonged to the globally disseminated sequence type ST131. ESBL-genes of the CTX-M-1 and CTX-M-9 phylogroups were identified in all ten isolates. The plasmid content (plasmidome) of each strain was analyzed using a combination of molecular methods and high-throughput sequencing. Hidden Markov Model-based analysis of unassembled sequencing reads was used to analyze the genetic diversity of the plasmid samples and to detect resistance genes. Each isolate contained between two and eight distinct plasmids, and at least 22 large plasmids were identified overall. The plasmids were variants of pUTI89, pKF3-70, pEK499, pKF3-140, pKF3-70, p1ESCUM, pEK204, pHK17a, p083CORR, R64, pLF82, pSFO157, and R721. In addition, small cryptic high copy-number plasmids were frequent, containing one to seven open reading frames per plasmid. Three clustered groups of such small cryptic plasmids could be distinguished based on sequence similarity. Extrachromosomal prophages were found in three isolates. Two of them resembled the E. coli P1 phage and one was previously unknown. The present study confirms plasmid multiplicity in multi-resistant E. coli. We conclude that high-throughput sequencing successfully provides information on the extrachromosomal gene content and can be used to generate a genetic fingerprint of possible use in epidemiology. This could be a valuable tool for tracing plasmids in outbreaks

Extended-spectrum beta-lactamase-producing Escherichia coli in patients with travellers' diarrhoea.

Abstract The identification of patients carrying extended-spectrum beta-lactamase (ESBL)-producing bacteria is important, since these patients are at risk of receiving inappropriate empirical therapy if they become infected. The purpose of this study was to investigate the occurrence of ESBL-producing bacteria in patients with travellers' diarrhoea. Patients with travellers' diarrhoea (N = 242) having delivered stool samples for the diagnosis of Salmonella, Shigella, Yersinia or Campylobacter, were also examined for ESBL-producing Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. The overall prevalence of faecal carriage of ESBL-producing bacteria was 24% (58/242). Of the patients who had travelled in Europe, 3% (2/63) were found to be ESBL carriers in comparison to 36% (50/138) of those who had travelled outside Europe. ESBL-producing E. coli was especially common among patients returning from India (11/14), Egypt (19/38; 50%) and Thailand (8/38; 22%). In total, 90% of the genes of the ESBL-positive samples were of CTX-M type. The CTX-M-1 group dominated, followed by the CTX-M-9 group. The repetitive sequence-based PCR fingerprint pattern showed that there was no similarity between the ESBL strains found. Patients who have travelled outside Europe are at high risk of being colonized with ESBL-producing Enterobacteriaceae, and, if infected, are also at risk of receiving inappropriate empirical antibiotic therapy

Plasmidome-Analysis of ESBL-Producing Escherichia coli Using Conventional Typing and High-Throughput Sequencing

Infections caused by Extended spectrum beta-lactamase (ESBL)-producing E. coli are an emerging global problem, threatening the effectiveness of the extensively used beta-lactam antibiotics. ESBL dissemination is facilitated by plasmids, transposons, and other mobile elements. We have characterized the plasmid content of ESBL-producing E. coli from human urinary tract infections. Ten diverse isolates were selected; they had unrelated pulsed-field gel electrophoresis (PFGE) types (<90% similarity), were from geographically dispersed locations and had diverging antibiotic resistance profiles. Three isolates belonged to the globally disseminated sequence type ST131. ESBL-genes of the CTX-M-1 and CTX-M-9 phylogroups were identified in all ten isolates. The plasmid content (plasmidome) of each strain was analyzed using a combination of molecular methods and high-throughput sequencing. Hidden Markov Model-based analysis of unassembled sequencing reads was used to analyze the genetic diversity of the plasmid samples and to detect resistance genes. Each isolate contained between two and eight distinct plasmids, and at least 22 large plasmids were identified overall. The plasmids were variants of pUTI89, pKF3-70, pEK499, pKF3-140, pKF3-70, p1ESCUM, pEK204, pHK17a, p083CORR, R64, pLF82, pSFO157, and R721. In addition, small cryptic high copy-number plasmids were frequent, containing one to seven open reading frames per plasmid. Three clustered groups of such small cryptic plasmids could be distinguished based on sequence similarity. Extrachromosomal prophages were found in three isolates. Two of them resembled the E. coli P1 phage and one was previously unknown. The present study confirms plasmid multiplicity in multi-resistant E. coli. We conclude that high-throughput sequencing successfully provides information on the extrachromosomal gene content and can be used to generate a genetic fingerprint of possible use in epidemiology. This could be a valuable tool for tracing plasmids in outbreaks

No. of <i>E. coli</i> (n = 320) isolates resistant to trimethoprim only and in combination with other antibiotics and in relation to the presence of integrons.

<p>TMP = trimethoprim, AMP = ampicillin, NAL = nalidixic acid, MEC = mecillinam, CFR = cefadroxil and NIT = nitrofurantoin.</p

The relationship between <i>dfr-</i>genes and integron class I and II in <i>E. coli</i> (n = 320) and <i>K. pneumoniae</i> (n = 54).

a<p>Including 10 isolates positive for both Igr1 and 2.</p>b<p>Including one isolate positive for both Igr 1 and 2.</p