Antimicrobial resistance of Escherichia coli isolates from outpatient urinary tract infections in women in six European countries including Russia

Objectives In the Northern Dimension Antibiotic Resistance Study (NoDARS), Finland, Germany, Latvia, Poland, Russia and Sweden collected urine samples from outpatient women (aged 18–65 years) with symptoms of uncomplicated urinary tract infection (UTI) to investigate the levels of antimicrobial resistance (AMR) among Escherichia coli isolates. Methods A total of 775 E. coli isolates from 1280 clinical urine samples were collected from October 2015 to January 2017. Antimicrobial susceptibility testing was performed and the results were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Results Overall AMR rates to the commonly used antibiotics nitrofurantoin, fosfomycin and mecillinam (except for Germany that was missing a result for mecillinam) were 1.2%, 1.3% and 4.1%, respectively. The highest overall resistance rates were determined for ampicillin (39.6%), trimethoprim (23.8%), trimethoprim/sulfamethoxazole (22.4%), amoxicillin/clavulanic acid (16.7%) and ciprofloxacin (15.1%), varying significantly between countries. The rate of extended-spectrum β-lactamase (ESBL) production was 8.7%. None of the isolates showed resistance to meropenem. Conclusions In most cases, low AMR rates were detected against the first-line antibiotics recommended in national UTI treatment guidelines, giving support to their future use. These results also support the European Association of Urology guidelines stating that nitrofurantoin, fosfomycin and mecillinam are viable treatment options for uncomplicated UTI.Peer Reviewe

Mycoplasma pneumoniae infections, 11 countries in Europe and Israel, 2011 to 2016

Background: Mycoplasma pneumoniae is a leading cause of community-acquired pneumonia, with large epidemics previously described to occur every 4 to 7 years. Aim: To better understand the diagnostic methods used to detect M. pneumoniae; to better understand M. pneumoniae testing and surveillance in use; to identify epidemics; to determine detection number per age group, age demographics for positive detections, concurrence of epidemics and annual peaks across geographical areas; and to determine the effect of geographical location on the timing of epidemics. Methods: A questionnaire was sent in May 2016 to Mycoplasma experts with national or regional responsibility within the ESCMID Study Group for Mycoplasma and Chlamydia Infections in 17 countries across Europe and Israel, retrospectively requesting details on M. pneumoniae-positive samples from January 2011 to April 2016. The Moving Epidemic Method was used to determine epidemic periods and effect of country latitude across the countries for the five periods under investigation. Results: Representatives from 12 countries provided data on M. pneumoniae infections, accounting for 95,666 positive samples. Two laboratories initiated routine macrolide resistance testing since 2013. Between 2011 and 2016, three epidemics were identified: 2011/12, 2014/15 and 2015/16. The distribution of patient ages for M. pneumoniae-positive samples showed three patterns. During epidemic years, an association between country latitude and calendar week when epidemic periods began was noted. Conclusions: An association between epidemics and latitude was observed. Differences were noted in the age distribution of positive cases and detection methods used and practice. A lack of macrolide resistance monitoring was noted

Regulation of virulence gene expression in Staphylococcus aureus

The pathogenic bacterium Staphylococcus aureus has the ability to cause a wide variety of human diseases, ranging from superficial abscesses and wound infections to deep and systemic infections such as osteomyelitis, endocarditis and septicaemia. The ability to cause disease has been attributed to a large number of toxins and digesting enzymes as well as to proteins at the bacterial surface that bind various host molecules. These so-called virulence factors are accessory, and are supposed to be synthesised in response to the specific needs during the course of the infectious process. The work described in this thesis aims at a better understanding of the mechanisms that regulate the expression of virulence factors. Two interacting regulatory systems, agr (accessory gene regulator) and sar (staphylococcal accessory regulator), are involved in this regulation. The agr locus, which encodes a two-component signal transduction system responding to cell density, controls the expression of at least 25 different virulence factors. The effector molecule of the agr system is a regulatory RNA molecule, named RNAIII. The sar locus has been shown to regulate several staphylococcal virulence genes by modulating the activity of agr, but also via agr-independent mechanisms. The effector of the sar locus is a 14.7 kDa DNA binding protein, SarA. In animal models of infection both agr and sarA have been shown to affect virulence. How RNAIII and sarA function at the molecular level is, however, poorly understood. The structure and function of the RNAIII promoter have been studied in detail showing that SarA, which regulates the synthesis of RNAIII under certain growth conditions, binds to multiple sites within the RNAIII promoter region. It has also been shown that a region of 93 bp upstream of the transcription start point is sufficient for agr-dependent regulation of RNAIII synthesis. The RNAIII genes of several coagulase negative staphylococcal species S. epidermidis, S. simulans and S. warneri have been identified and analysed. The RNAIII molecules from the coagulase negative staphylococci were able to partially complement an RNAIII deficient S. aureus mutant. By the construction of hybrid RNAIII molecules it has also been demonstrated that highly conserved primary and secondary structures in both the 5´- and the 3´-half of the RNAIII molecule are required for regulation of virulence genes, and that separate parts of the molecule were involved in regulation of different target genes. Several genes known to be regulated by RNAIII have been demonstrated to be regulated by the sarA locus, independent of its effect on expression of RNAIII. By electrophoresis mobility shift experiments and DNase footprinting, SarA has been found to bind in a very similar way to the promoter regions of genes that are either activated or repressed by sarA. SarA does not appear to recognise a conserved DNA sequence motif but rather binds to AT-rich sequences. New potential regulators of agr (RNAIII), hla (alpha-hemolysin), ssp (serine protease) and spa (protein A) have been searched for using specific promoter DNA linked to magnetic beads. Of several new candidate regulators, one protein with a high degree of similarity to SarA, named SarH1 (Sar Homologue 1) has been characterised and found to be part of the agr-sarA regulatory network controlling virulence gene expression. By computer searches in the unfinished S. aureus genome databases four additional Sar homologues have been found, some of which may also be involved in this regulatory network

SarA Is a Repressor of hla (α-Hemolysin) Transcription in Staphylococcus aureus: Its Apparent Role as an Activator of hla in the Prototype Strain NCTC 8325 Depends on Reduced Expression of sarS

In most Staphylococcus aureus strains, inactivation of sarA increases hla transcription, indicating that sarA is a repressor. However, in S. aureus NCTC 8325 and its derivatives, used for most studies of hla regulation, inactivation of sarA resulted in decreased hla transcription. The disparate phenotype of strain NCTC 8325 seems to be associated with its rsbU mutation, which leads to σ(B) deficiency. This has now been verified by the demonstration that sarA repressed hla transcription in an rsbU(+) derivative of strain 8325-4 (SH1000). That sarA could act as a repressor of hla in an 8325-4 background was confirmed by the observation that inactivation of sarA in an agr sarS rot triple mutant dramatically increased hla transcription to wild-type levels. However, the apparent role of sarA as an activator of hla in 8325-4 was not a result of the rsbU mutation alone, as inactivation of sarA in another rsbU mutant, strain V8, led to increased hla transcription. Northern blot analysis revealed much higher levels of sarS mRNA in strain V8 than in 8325-4, which was likely due to the mutation in the sarS activator, tcaR, in 8325-4, which was not found in strain V8. On the other hand, the relative increase in sarS transcription upon the inactivation of sarA was 15-fold higher in 8325-4 than in strain V8. Because of this, inactivation of sarA in 8325-4 means a net increase in repressor activity, whereas in strain V8, inactivation of sarA means a net decrease in repressor activity and, therefore, enhanced hla transcription

Senior Preschoolers Moral Value Formation in Understanding by Getting Acquaintanced to Literary Works

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

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

Development of a real-time SYBRGreen PCR assay for rapid detection of acquired AmpC in Enterobacteriaceae

Introduction: Acquired AmpC enzymes, classified as miscellaneous extended-spectrum beta-lactamase (ESBLM) enzymes according to a recently proposed beta-lactamase classification, are increasing according to several publications. Simple and rapid methods for detection of ESBLM are needed for appropriate infection control. A gel-based multiplex PCR method for acquired bla(AmpC) detection and subtype classification has been available for several years. Here, we describe a modification of the protocol to suit real-time PCR platforms and to include novel genotypes. Material and methods: Clinical isolates with clavulanic acid non-reversible non-susceptibility to extended-spectrum cephalosporins were subjected to combination disk testing with cefoxitin +/- cloxacillin at Malmo University Hospital. Phenotypical AmpC production was defined as cloxacillin reversible cefoxitin resistance. In this study 51 phenotypical AmpC-producing isolates, were subjected to the acquired bla(AmpC) real-time PCR assay. The acquired blaAmpC positive isolates were further characterized by DNA sequencing of the acquired AmpC encoding gene, Pulsed-Field Gel Electrophoresis (PFGE) and PCR-based replicon typing. Results and discussion: The real-time PCR assay was able to detect and sub-classify all acquired bla(AmpC) genes described to date. The assay can be performed in less than 3 h, including pre-PCR preparations. Analysis of the isolate collection resulted in 18 of 51 phenotypical AmpC-producing isolates being positive in the acquired bla(AmpC) real-time multiplex PCR assay; 17 of subtype CIT and one DHA. Sequence analysis identified 16 isolates as blaCMY-2, one as blaCMY-16 and one as blaDHA-1. Detected plasmid replicon types were I1 and B/O. Two of the E. coli isolates were identical according to PFGE and the others were unrelated. (C) 2010 Elsevier B.V. All rights reserved

Domain analysis of sequencing reads using pfam profile Hidden Markov Models.

<p>(A) Stacked bar-plot showing the number of 454 sequencing reads with hits to pfam models (E-value<1e–05). Turquoise and red colours refer to no hits and hits, respectively. Sequencing reads were conceptually translated to all six reading frames prior to searching. (B) Heat-map analysis of the domain content in the isolates (942 domains were found). The horizontal axis shows the pfam domain and the y-axis indicates the isolate. The unique pfam domain identifier is given on the horizontal axis. The colour intensity is logarithmically related to the number of sequencing reads matching the domain. The white colour indicates few reads matching a domain and colour transition to red indicates that more reads were found. Isolates are clustered after similarity. The bottom-right table indicates the number of non-redundant pfam domains per isolate; i.e., counting each domain only once. (*) The sample has likely been affected by phage-mediated DNA-degradation.</p

Distribution and properties of small cryptic plasmids.

<p>(A) Illustration of the content of small cryptic plasmids in the isolates. Circles represent plasmids and the plasmid size is shown on the y-axis in kilobases and the x-axis shows the sample ECO- identifier. (B) Neighbour-joining tree of small cryptic plasmids indicating their sequence-based relationships. Sequences were aligned with ClustalW v.2.1 and the tree was created with MEGA v.5 (default settings) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065793#pone.0065793-Tamura1" target="_blank">[35]</a>. Scale bar = substitutions per site. Support values are indicated at branches. (C and D) Sequence similarity blocks between small cryptic plasmids (violet stripes). Arrows indicate ORFs longer than 300 bp. Similarity blocks were identified with Mauve v.2.3.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065793#pone.0065793-Darling1" target="_blank">[36]</a>, and plotted with genoPlotR v.0.8.1. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065793#pone.0065793-Guy1" target="_blank">[37]</a>. ARP = putative aminoglycoside resistance protein; T2DM = putative type II DNA- methyltransferase; RE = putative EcoVIII restriction endonuclease.</p

Plasmid characteristics and content.

a<p>Average 454 coverage is specified within parenthesis.</p><p>Abbreviations: (nd) not detected; (na) not applicable; and ∑ sum of unassigned contigs.</p