phiD12-Like Livestock-Associated Prophages Are Associated With Novel Subpopulations of Streptococcus agalactiae Infecting Neonates

Group B Streptococcus (GBS) is a major cause of invasive disease in neonates worldwide. Monitoring data have revealed a continuing trend toward an increase in neonatal GBS infections, despite the introduction of preventive measures. We investigated this trend, by performing the first ever characterization of the prophage content for 106 GBS strains causing neonatal infections between 2002 and 2018. We determined whether the genome of each strain harbored prophages, and identified the insertion site of each of the prophages identified. We found that 71.7% of the strains carried at least one prophage, and that prophages genetically similar to livestock-associated phiD12, carrying genes potentially involved in GBS pathogenesis (e.g., genes encoding putative virulence factors and factors involved in biofilm formation, bacterial persistence, or adaptation to challenging environments) predominated. The phiD12-like prophages were (1) associated with CC17 and 1 strains (p = 0.002), (2) more frequent among strains recovered during the 2011–2018 period than among those from 2002–2010 (p < 0.001), and (3) located at two major insertion sites close to bacterial genes involved in host adaptation and colonization. Our data provide evidence for a recent increase in lysogeny in GBS, characterized by the acquisition, within the genome, of genetic features typical of animal-associated mobile genetic elements by GBS strains causing neonatal infection. We suggest that lysogeny and phiD12-like prophage genetic elements may have conferred an advantage on GBS strains for adaptation to or colonization of the maternal vaginal tract, or for pathogenicity, and that these factors are currently playing a key role in the increasing ability of GBS strains to infect neonates

Methicillin-Susceptible ST398 Staphylococcus aureus Responsible for Bloodstream Infections: An Emerging Human-Adapted Subclone?

In the course of an annual 3-month bloodstream infections (BSI) survey conducted during a four-year period in 31 healthcare institutions located in three noncontiguous French regions, we report 18 ST398 Staphylococcus aureus BSI. ST398 BSI incidence showed a seven-fold increase during the study period (0.002 per 1,000 patient days in 2007 vs. 0.014 in 2010). ST398 BSI isolates differed from the pig-borne multiresistant clone: 17/18 BSI isolates were methicillin susceptible and none was of t011, t034 or t108 pig-borne spa-types. ST398 BSI isolates had homogenous resistance patterns (15/18 with only Eryr) and prophagic content (all harboured the hlb-converting Sau3int phage). The clustering of BSI and pig-borne isolates by spa-typing and MLVA, the occurrence of Sau3int phage in BSI isolates and the lack of this phage in pig-borne isolates suggest that the emergence of BSI isolates could have arisen from horizontal transfer, at least of the Sau3int phage, in genetically diverse MSSA ST398 isolates. The acquisition of the phage likely plays a role in the increasing ability of the lysogenic ST398 isolates to colonize human. The mode of acquisition of the non pig-borne ST398 isolates by our 18 patients remains unclear. ST398 BSI were diagnosed in patients lacking livestock exposure and were significantly associated with digestive portals of entry (3/18 [16.7%] for ST398 vs. 19/767 [2.5%] for non ST398 BSI; p = .012). This raises the question of possible foodborne human infections. We suggest the need for active surveillance to study and control the spread of this human-adapted subclone increasingly isolated in the hospital setting

Efficacy and safety of a two-step method of skin preparation for peripheral intravenous catheter insertion: a prospective multi-centre randomised trial

We have developed a two-step procedure for preparing the skin before peripheral venous catheter (PVC) insertions. This procedure involves two successive swabbings with wipes soaked in alcoholic antiseptic. We investigated whether this two-step procedure was as effective and safe as the standard four-step procedure – washing with detergent, rinsing, drying, applying antiseptic – by carrying out a multicentre randomised equivalence study comparing the frequency of precursor signs of infection at the site of insertion for the two skin preparation procedures. The study was carried out over an eight-month period, and 248 PVC insertion sites were evaluated. The two-step procedure was used for 130 subjects and the standard procedure for 118. Taking into account all the confounding factors predisposing patients to the complications studied, the characteristics of the two groups of patients were found to be similar, with no significant differences noted. The incidence of precursor signs of infection was 11 % 24 hours after PVC insertion (27/248), 25 % at 48 hours (50/203) and at 29 % at 72 hours (34/119). Eleven patients had complications necessitating the withdrawal of the PVC: sensitivity of the insertion site, with redness and/or slight swelling and/or a palpable venous cord. No major complications were observed in this study. The frequency of local complications associated with PVCs reported in this study, whether simple or severe, was not affected by the skin preparation procedure used for PVC insertion (two-step or four-step procedure)

CC9 Livestock-Associated Staphylococcus aureus Emerges in Bloodstream Infections in French Patients Unconnected With Animal Farming

We report 4 bloodstream infections associated with CC9 agr type II Staphylococcus aureus in individuals without animal exposure. We demonstrate, by microarray analysis, the presence of egc cluster, fnbA, cap operon, lukS, set2, set12, splE, splD, sak, epiD, and can, genomic features associated with a high virulence potential in human

Evaluation of the Swabbing of Disposable Absorbent Incontinence Products for Assessing the Carriage of Multiresistant Enterobacteriaceae in Nursing Home Residents

We compared the performance of incontinence product (IP) and rectal swabbing for the detection of multidrug-resistant Enterobacteriaceae (MDRE) carriage in a large multicenter study conducted in February 2017 among the residents of 23 French nursing homes. The study included 547 residents who habitually wore IP, 88 of whom were MDRE carriers (16.1%). Positive results were obtained for both rectal and IP swabs for 64 of these residents, for rectal swabs only for 22 and for IP swabs only for two of these patients. The estimated prevalence of MDRE carriage depended on the type of sample: 15.7% for rectal swabs and 12.1% for IP swabs (p < 0.001). The positive percent agreement was 84.2% and the negative percent agreement was 97.4%. Rectal swabbing remains the best method for detecting MDRE carriage in elderly residents, but our findings provide support for the use of swabs from IP used overnight to increase response rates in MDRE surveys in elderly residents that habitually wear IP, when rectal swabbing is not feasible

Facteurs contextuels d’efficacité des dispositifs de prévention des infections associées aux soins : les apports de la recherche

In hospital, the number of deaths involving healthcare-associated infection (HAI) remains high, despite the numerous control approaches used. In 2015, Zingg et al. asked for a multifactorial and context-sensitive approach to HAI prevention. This led us to undertake a synthesis of current research work, looking for context-sensitive factors liable to impact the effectiveness of prevention approaches. We identified three types of factors: organisational, human, and patient-related. We suggest that an innovative methodology could be used for an improved coverage of the complexity of HAI prevention. The potential advantages of this approach are illustrated by the results of a preliminary test conducted on the data collected during the Spiadi mission. This was part of a research project initiated in a university research institute dedicated to prevention.Le nombre de décès relatifs à des infections associées aux soins (IAS) dans les hôpitaux reste élevé malgré les nombreux dispositifs de prévention mis en place. Nous répondons à l’appel de Zingg et al. en 2015 d’une approche multifactorielle et contextuelle de prévention des IAS. Nous réalisons pour cela une synthèse des recherches sur les facteurs contextuels qui peuvent influencer l’efficacité des dispositifs de prévention. Nous identifions ainsi des facteurs à trois niveaux : organisationnel, humain et relatif au patient. Nous suggérons, en outre, qu’adopter une méthodologie innovante, l’approche « en configurations de facteurs » pourrait permettre de mieux embrasser la complexité de la prévention des IAS. Nous illustrons l’intérêt potentiel de cette approche à travers les résultats d’un test préliminaire effectué sur des données de la mission de surveillance et prévention des infections associées aux dispositifs invasifs (Spiadi) obtenues dans le cadre d’un projet de recherche initié au sein d’un institut universitaire de recherche sur la prévention

Facteurs contextuels d’efficacité des dispositifs de prévention des infections associées aux soins : les apports de la recherche

In hospital, the number of deaths involving healthcare-associated infection (HAI) remains high, despite the numerous control approaches used. In 2015, Zingg et al. asked for a multifactorial and context-sensitive approach to HAI prevention. This led us to undertake a synthesis of current research work, looking for context-sensitive factors liable to impact the effectiveness of prevention approaches. We identified three types of factors: organisational, human, and patient-related. We suggest that an innovative methodology could be used for an improved coverage of the complexity of HAI prevention. The potential advantages of this approach are illustrated by the results of a preliminary test conducted on the data collected during the Spiadi mission. This was part of a research project initiated in a university research institute dedicated to prevention.Le nombre de décès relatifs à des infections associées aux soins (IAS) dans les hôpitaux reste élevé malgré les nombreux dispositifs de prévention mis en place. Nous répondons à l’appel de Zingg et al. en 2015 d’une approche multifactorielle et contextuelle de prévention des IAS. Nous réalisons pour cela une synthèse des recherches sur les facteurs contextuels qui peuvent influencer l’efficacité des dispositifs de prévention. Nous identifions ainsi des facteurs à trois niveaux : organisationnel, humain et relatif au patient. Nous suggérons, en outre, qu’adopter une méthodologie innovante, l’approche « en configurations de facteurs » pourrait permettre de mieux embrasser la complexité de la prévention des IAS. Nous illustrons l’intérêt potentiel de cette approche à travers les résultats d’un test préliminaire effectué sur des données de la mission de surveillance et prévention des infections associées aux dispositifs invasifs (Spiadi) obtenues dans le cadre d’un projet de recherche initié au sein d’un institut universitaire de recherche sur la prévention

Ligation Chain Reaction-Rolling Circle Amplification for Single Nucleotide Polymorphism Genotyping

連結-滾動循環擴增技術是一種運用在核酸分析上結合扣鎖式探針連結酶鏈反應及多分枝型滾動循環擴增的分析技術。一般設計的扣鎖式探針由於此偵測引子的存在而使得探針的長度大多在於100個核苷酸以上。本研究中利用重疊序列設計以縮減探針長度，設計出一段長64個核酸的探針用以分析MTHFR c677t多型性。在設計上是利用扣鎖式探針的兩端與MTHFR c677t序列互補故可辨識並與目標序列結合，當探針與目標序列結合後，連結酶可將探針頭尾連結形成一環狀探針，而當探針無法互補結合時，連接酶無法作用使探針依舊是線狀，存在環狀探針時，滾動放大的結果可使目標核酸序列的訊號在肉眼下簡單判讀。在結果上，利用SYBR Green I及即時偵測系統偵測可有效對MTHFR基因進行單一核苷酸分型。Rolling circle amplification (RCA) of circularizing oligo nucleotide probes (padlock probes) is a sensitive method for short DNA fragment detection and amplification. Although oligonucleotide can be chemically synthesized, the longer probe sequences will have a lower synthesis yield and sequence accuracy. Here we designed a 64nt padlock probe for MTHFR c677t polymorphism detection. The shorter probe sequence makes it easy to obtain from chemical synthesis. In this study, the both ends of padlock probes are designed complementary to MTHFR c677t sequence. With a sensitive DNA ligase, the matched probe will be ligated and form a circular-form probe. Otherwise, the mismatched probe fails to be ligated and keeps in a liner form. The following hyper-branched rolling circle amplification of circular-form probe makes it easy to analyze the probe ligation result. RCA method detection sensitivity and specificity is based on probe ligation stage, which becomes a critical step in RCA detection. The ligase chain reaction method can help to increase ligation efficiency and more linked circle-form padlock probe yield for paired sequence. We amplified the circular-form padlock probe with detection primer and isothermal polymerase. The isothermal polymerase character makes RCA can be a ready-to-use detection method for study.中文摘要 i ABSTRACT ii 目錄 iii 表目錄 vi 圖目錄 vii 附錄目錄 viii 第一章 緒論 1 第一節 滾動循環增幅(Rolling circle amplification,RCA) 1 第二節 單核苷酸多型性(Single nucleotide polymorphism,SNP) 3 第三節 甲基四氫葉酸還原酶 (Methylenetetrahydrofolate reductase,MTHFR) 基因 5 第四節 點突變檢測法簡介 8 一、Single-strand conformation polymorphism (SSCP) 8 二、Real-time PCR 8 三、Denaturing high performance liquid chromatography (dHPLC) 9 四、Restriction fragment length polymorphism(RFLP) 9 五、Direct DNA sequencing 10 第五節 研究動機 12 第二章 材料與方法 14 第一節 材料 14 一、Genomice DNA 14 二、寡核苷酸(Oligo nucleotides) 14 三、儀器 15 第二節 方法 15 一、PCR 15 二、不對稱PCR (Asymmetric PCR,As-PCR) 16 三、連接酶鏈反應(ligase chain reaction, LCR) 17 四、Exonuclease I digestion 18 五、超分支滾動循環擴增(hyperbranched rolling circle amplification,HRCA) 18 六、瓊脂凝膠電泳(agarose gel electrophoresis) 20 第三章 結果 21 一、比較PCR與不對稱PCR(Asymmetric PCR)差異 21 二、扣鎖探針設計(Padlock probe design) 21 三、連接酶鏈反應(ligation chain reaction,LCR) 23 四、滾動循環增幅(Rolling circle amplification,RCA)與超分支滾動循環增幅(hyperbranched RCA,HRCA) 23 五、Bst DNA聚合酶RCA放大 24 六、利用本法做基因分型驗證 25 第四章 結論 26 第五章 討論 27 參考文獻 31 表 33 圖 36 附錄 4

Analysis of the prophages carried by human infecting isolates provides new insight into the evolution of Group B Streptococcus species

International audienceObjectives: Group B Streptococcus (GBS) emerged in the 1970s as a major cause of neonatal infections, and has been increasingly associated with infections in adults since the 1990s. Prophages have been suspected to have driven these epidemiological trends. We have characterized the prophages harboured by 275 human GBS isolates belonging to the major lineages.& para;& para;Methods: We applied whole genome sequencing (WGS) to 14 isolates representative of the diversity within GBS species, located and identified their prophages. Using prediction tools, we searched for prophage elements potentially involved with the ability of GBS to infect humans. Using the data obtained by WGS, we designed a PCR-based tool and studied the prophage content of 275 isolates.& para;& para;Results: WGS of the 14 isolates revealed 22 prophages (i) distributed into six groups (A-F), (ii) similar to phages and prophages from GBS and non-GBS streptococci recovered from livestock, and (iii) carrying genes encoding factors previously associated with host adaptation and virulence. PCR-based detection of prophages revealed the presence of at least one prophage in 72.4% of the 275 isolates and a significant association between neonatal infecting isolates and prophages C, and between adult infecting isolates and prophages A.& para;& para;Conclusions: Our results suggest that prophages (possibly animal-associated) have conditioned bacterial adaptation and ability to cause infections in neonates and adults, and support a role of lysogeny with the emergence of GBS as a pathogen in human. (C) 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved

<em>Helicobacter caesarodunum</em>: A novel human pathogen identified in a bloodstream infection

National audienc