The authors would like to thank Trevor Lawley at the Wellcome Trust Sanger Institute for C. difficile R20291 spores and James S. Guy at the North Carolina State University College of Veterinary Medicine for Vero cells, both utilized in this manuscript. Animal histopathology was performed in the LCCC Animal Histopathology Core Facility at the University of North Carolina at Chapel Hill, with special assistance from Traci Raley and Amanda Brown. The LCCC Animal Histopathology Core is supported in part by an NCI Center Core Support Grant (2P30CA016086-40) to the UNC Lineberger Comprehensive Cancer Center. We would also like to thank Vincent Young, Anna Seekatz, Jhansi Leslie, and Cassie Schumacher for helpful discussions on the Vero cell cytotoxicity assay protocol. JAW is funded by the Ruth L. Kirschstein National Research Service Award Research Training grant T32OD011130 by NIH. CMT is funded by the career development award in metabolomics grant K01GM109236 by the NIGMS of the NIH.

<ol>
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The authors have nothing to disclose at this time.

This protocol characterizes the clinical course, including weight loss, bacterial colonization, toxin cytotoxicity, and histopathological changes in the gastrointestinal tract, of antibiotic-treated mice challenged with C. difficile R20291 spores. There are several critical steps within the protocol where attention to detail is essential. Accurate calculation of the C. difficile spore inoculum is critical. This calculation is based on the original C. difficile spore stock enumeration, which should be consistent over multiple spore enumerations prior to starting this protocol. If the inoculum calculation is erroneous, it will result in an inaccurate inoculation dose of the mice. On the same note, preparation and dilutions of C. difficile spores based on these calculations should be prepared with care. Any dilution errors will result in an inaccurate starting spore inoculum. Lastly, precautions should be taken to avoid contamination of antibiotic-treated control mice (not inoculated with C. difficile spores). Cefoperazone-treated mice are extremely susceptible to C. difficile colonization and thus should not be in direct contact with C. difficile-infected mice28-30. If the aforementioned critical steps are recognized, successful execution of the CDI mouse model is expected. 
Modification of the dose or strain of C. difficile spores administered to the mice may alter the outcomes of this model. Increasing the spore inoculum administered to mice could increase the morbidity and mortality observed, depending on the strain of C. difficile administered. A dose response experiment, evaluating a range of several inoculum doses, is recommended if using other C. difficile strains in order to characterize the clinical course of CDI. 
This protocol uses TCCFA medium for C. difficile enumeration of spores and vegetative cells collectively. However, if distinct phases (such as germination and outgrowth) of the C. difficile lifecycle are desired, differential plating is required. Non-heat-treated contents, as described in this protocol, will yield the CFU per g content of spores and vegetative cells together. If the same GI content is heat-treated (20 min at 65 &#176;C) and then plated on TCCFA media, this will yield a CFU of spores only, since C. difficile vegetative cells are destroyed by heat. Comparison of C. difficile enumeration using these two plating techniques allows for more detailed information about the C. difficile lifecycle in vivo. 
One major limitation of this mouse model is that the results may not be reproducible in mice from different strains or vendors. Recall that disruption of the gut microbiota following cefoperazone treatment renders mice susceptible to CDI28. Variations in the initial composition of the gut microbiota between different murine strains from different vendors could alter the mouse model of CDI31. Once researchers have successfully completed this model, it is recommended that the mouse strain and vendor from where the mice are acquired stay consistent for additional studies to limit variations in the gut microbiota composition, which could alter the outcomes of the model. 
Another limitation of the protocol is that the Vero cell cytotoxicity assay does not quantitatively measure C. difficile toxin levels in vivo. Instead, epithelial (Vero) cell cytotoxicity represents a qualitative approach that measures relative cytotoxicity of C. difficile toxins present in samples. Other techniques such as qRT-PCR and immunoblotting would be helpful to determine the amount of toxin present in the samples. 
Various models of CDI are currently being used, including the Syrian hamster model, ex vivo models, and human intestinal organoid models13,14,30,32. However, the mouse model of CDI provides the increased availability of commercially-available murine reagents and assays to further examine the pathogenesis of C. difficile. Other mouse models of CDI have used different strains of C. difficile, such as VPI 10463, which results in substantial mortality and thus does not recapitulate human CDI12,17,29. In this model, C. difficile R20291 was selected for its genetic tractability and clinical relevance as a recent 027 epidemic strain33,34. Compared to other murine models of CDI, R20291 was not uniformly lethal at the dose administered, allowing for observation of a prolonged clinical course of infection concordant with human disease. Overall, this mouse model proves to be a valuable experimental platform of CDI. This fully-characterized C. difficile mouse model can be utilized to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Clostridium difficile è un anaerobica,, bacillo spore gram-positivi che causa pericolo di vita la diarrea 1. Infezione da C. difficile (CDI) è associato ad un aumento della morbilità e della mortalità umana e si traduce in più di 4,8 miliardi di $ in spese sanitarie per anno 1-4. Nel 2013, i Centri per il Controllo e la Prevenzione delle Malattie classificati C. difficile come un rischio resistenza agli antibiotici urgente, indicando che essa pone una minaccia urgente per la salute pubblica 1. Attualmente, il trattamento antibiotico con vancomicina e metronidazolo sono considerati lo standard di cura per CDI 5. Purtroppo, il ripetersi di CDI dopo il successo del trattamento con antibiotici è alto, che si verificano nel 20 - 30% dei pazienti 2,5-7. Pertanto, la scoperta di nuove terapie contro questo patogeno enterico è necessario. Per valutare terapie contro il C. difficile, un modello animale approssimare la malattia umana in acè necessario un ceppo linically rilevanti. Inizialmente, i postulati di Koch sono stati stabiliti per il C. difficile nel 1977 utilizzando un modello di criceto siriano clindamicina trattati con 8. Questo modello è ancora utilizzato oggi per studiare gli effetti delle tossine di C. difficile sulla patogenesi 9,10. Tuttavia, CDI nel modello criceto si traduce in alti tassi di mortalità e non, sul ravvicinamento delle manifestazioni della malattia insidiosi croniche che possono essere visti in esseri umani con CDI 10,11. Sulla base della accessibilità e la disponibilità di piattaforme reagente murini in ricerca, un modello murino di CDI è rilevante. Nel 2008, un modello di topo robusto CDI è stato istituito con il trattamento di topi con un cocktail di antibiotici nell&#39;acqua potabile (kanamicina, gentamicina, colistina, metronidazolo e vancomicina) per 3 giorni, seguiti da un&#39;iniezione intraperitoneale di clindamicina 12. Questo topi resi sensibili alla colite CDI e grave. Dipenderezione della dose somministrata inoculo, una serie di segni clinici e letalità può osservare utilizzando questo modello. Da quel momento, i vari regimi antibiotici sono stati studiati che alterano la flora intestinale murino, diminuendo la resistenza colonizzazione al punto in cui C. difficile può colonizzare il tratto gastrointestinale (rivisto nel miglior et al. E Lawley &amp; Young) 13,14. Più di recente, un ampio spettro di cefalosporina, cefoperazone, data in acqua potabile per 5 o 10 giorni rende riproducibile topi suscettibili di CDI 15. Dal momento che la somministrazione di cefalosporine di terza generazione sono associati ad un aumentato rischio di CDI negli esseri umani, l&#39;uso del modello cefoperazone riflette in modo più accurato la malattia 16 in natura. Cefoperazone trattati topi suscettibili di C. difficile è stato messo in discussione con entrambe le spore di C. difficile e le cellule vegetativa di una varietà di ceppi che vanno a clinicarilevanza e virulenza 17. Nonostante alcuni degli studi originali che utilizzano C. cellule vegetative difficile del come la forma infettiva, spore di C. difficile del sono considerati la principale modalità di trasmissione 18. Negli ultimi dieci anni, C. difficile R20291, un NAP1 / BI / 027 ceppo, è emerso, causando epidemie di CDI 19,20. Abbiamo cercato di determinare il decorso clinico della malattia, quando i topi Cefoperazone trattati si sono sfidati con il C. difficile ceppo clinicamente rilevante e geneticamente trattabili, R20291. Dettagli Questo protocollo il decorso clinico, tra cui la perdita di peso, la colonizzazione batterica, la tossina citotossicità, e cambiamenti istopatologici nel tratto gastrointestinale dei topi sfidati con spore di C. difficile R20291. Nel complesso, questo modello di topo si rivela essere una piattaforma sperimentale prezioso per CDI approssimare malattie umane. Questo modello caratterizzato mouse può quindi essere utilizzato per valutare gli effettidi nuove terapie per il miglioramento della malattia clinica e sul restauro della resistenza colonizzazione contro C. difficile.

<table border="0" cellpadding="0" cellspacing="0" width="1037">
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		<col />
		<col />
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">#62 Perisept Sporidicial Disinfectant Cleaner&#160;</td>
			<td style="width:143px;">SSS Navigator</td>
			<td style="width:119px;">48027</td>
			<td style="width:483px;">This product will require dilution as recommended by the manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22 &#956;m filter</td>
			<td>Fisherbrand</td>
			<td>09-720-3</td>
			<td>Alternative to filter plate for indivdiual samples tested in the Vero Cell Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Needs to be heated in water bath at 37C prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.4% Trypan Blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1% Peniciilin/Streptomycin</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% heat inactivated FBS</td>
			<td>Gibco</td>
			<td>16140-071</td>
			<td>Needs to be heated in water bath at 37C prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1ml plastic syringe&#160;</td>
			<td>BD Medical Supplies</td>
			<td>309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 ml Micro Centrifuge Screw Cap</td>
			<td>Corning</td>
			<td>430917</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well cell culture flat bottom plate</td>
			<td>Costar Corning</td>
			<td>CL3595</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well filter plate</td>
			<td>Millipore</td>
			<td>MSGVS2210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesive Seal</td>
			<td>ThermoScientific</td>
			<td>AB-0558</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Bacto Agar</td>
			<td style="width:143px;">Becton Dickinson</td>
			<td style="width:119px;">214010</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Bacto Proteose Peptone</td>
			<td style="width:143px;">Becton Dickinson</td>
			<td style="width:119px;">211684</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefoperazone</td>
			<td>MP Bioworks</td>
			<td>199695</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cefoxitine</td>
			<td>Sigma</td>
			<td>C47856</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clostridium difficile Antitoxin Kit</td>
			<td>Tech Labs</td>
			<td>T5000</td>
			<td>Used as control for Vero Cell Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clostridium difficile Toxin A</td>
			<td>List Biological Labs</td>
			<td>152C</td>
			<td>Positive control for Vero Cell Assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-cycloserine</td>
			<td>Sigma</td>
			<td>C6880</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Distilled Water</td>
			<td>Gibco</td>
			<td>15230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM 1X Media</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>Needs to be heated in water bath at 37C prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fructose</td>
			<td>Fisher</td>
			<td>L95500</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td>Bright-Line, Sigma</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">KH2PO4</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">P285-500</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">MgSO4 (anhydrous)</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">M2643</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Millex-GS 0.22 &#956;m filter</td>
			<td style="width:143px;">Millex-GS</td>
			<td style="width:119px;">SLGS033SS</td>
			<td style="width:483px;">Filter for TCCFA plates&#160;</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:292px;">Na2HPO4</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">S-0876</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">S640-3</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Number 10 disposable scalpel blade</td>
			<td>Miltex, Inc</td>
			<td>4-410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Plates</td>
			<td>Fisherbrand</td>
			<td>14230244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic petri dish</td>
			<td>Kord-Valmark Brand</td>
			<td>2900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile plastic L-shaped cell spreader</td>
			<td>Fisherbrand</td>
			<td>14-665-230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe Stepper</td>
			<td>Dymax Corporation</td>
			<td>T15469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurocholate</td>
			<td>Sigma</td>
			<td>T4009</td>
			<td>Part of TCCFA plates (see below)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:292px;">C57BL/6J Mice</td>
			<td style="width:143px;">The Jackson Laboratory</td>
			<td align="right" style="width:119px;">664</td>
			<td style="width:483px;">Mice should be 5-8 weeks of age</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Olympus BX43F light microscope</td>
			<td style="width:143px;">Olympus Life Science</td>
			<td style="width:119px;"></td>
			<td style="width:483px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DP27 camera</td>
			<td style="width:143px;">Olympus Life Science</td>
			<td style="width:119px;"></td>
			<td style="width:483px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">cellSens Dimension software&#160;</td>
			<td style="width:143px;">Olympus Life Science</td>
			<td style="width:119px;"></td>
			<td style="width:483px;"></td>
		</tr>
	</tbody>
</table>

cefoperazone-treated mouse model of clinically-relevant clostridium difficile strain r20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Durante uno studio rappresentativo, 5 settimane di età C57BL / 6 topi WT sono stati pretrattati con cefoperazone nella loro acqua potabile (0,5 mg / ml) per 5 giorni e ha permesso a 2 giorni lavare con acqua potabile normale. I topi sono stati sfidati con 10 5 spore di C. difficile R20291 tramite sonda gastrica al giorno 0 (Figura 1A). I topi sono stati monitorati per la perdita di peso e segni clinici (letargia, inappetenza, diarrea, e la postura ingobbita) di CDI per 14 giorni. La sfida di C57BL / 6 topi WT con spore di C. difficile R20291 provocato diarrea entro 24 ore e significativa perdita di peso entro 48 ore post-sfida (Figura 1B). Anche se non osservata in questo esperimento, i topi sfidato con una dose maggiore di C. difficile R20291 possono diventare gravemente ammalati o avere superiore al 20% la perdita di peso da 48-72 ore post-sfida, che richiede quindi l&#39;eutanasia umana. Pertanto, topi richiedono un minimo di due volte al giornoil monitoraggio dopo sfida con spore di C. difficile. In questo studio rappresentativo, una notevole quantità di perdita di peso e sintomi clinici della malattia nei topi è stato osservato anche nei giorni 2-7 post-sfida (Figura 1B). A 7 giorni post-challenge, i topi hanno cominciato ad aumentare di peso, e segni clinici di malattia placata. Con l&#39;ultima settimana di questo esperimento, i topi è apparso clinicamente normale, senza evidenza dei segni clinici di CDI, tra cui la diarrea. Pellet fecali sono stati raccolti prima della sfida con spore di C. difficile e ogni 48 ore post-sfida (Figura 1C). Prima del trattamento antibiotico, topi non furono colonizzate con C. difficile. All&#39;interno di posta sfida 24 ore con spore di C. difficile, i topi sono stati colonizzati con 10 7 UFC di C. difficile per g di feci (Figura 1C &lt;/ Strong&gt;). Mice è rimasto persistentemente colonizzato da C. difficile tutto l&#39;esperimento, nonostante nessuna prova di perdita di peso o segni clinici di CDI per gli ultimi 7 giorni dell&#39;esperimento. <img alt="Figura 1" src="/files/ftp_upload/54850/54850fig1.jpg" /> Figura 1: Cefoperazone topi trattati sfidati con C. difficile R20291 perdita Exhibit peso e sono colonizzate con C. difficile. A) 5 settimane di età C57BL 6 topi / WT JAX (n = 3M / 3F per gruppo) sono stati pretrattati con cefoperazone nella loro acqua potabile (0,5 mg / ml) per 5 giorni e ha permesso a 2 giorni lavare con regolare potabile acqua. I topi sono stati sfidati con 10 5 spore di C. difficile R20291 tramite sonda gastrica il giorno 0. I topi sono stati monitorati per la perdita di peso e segni clinici (letargia, inappetenza, diarrea, e la postura ingobbita) di CDI per 14 giorni. Feci sono stati raccolti e utilizzati per il conteggio dei batteri prima di iniziare cefoperazone, subito after finire l&#39;antibiotico, e ai giorni 0, 1, 2, 3, 4, 5, 7, 9, 11, 13, e 14 durante l&#39;infezione. Necroscopia è stato eseguito nei giorni 0, 2, 4, 7, e 14 B) I topi ha perso una quantità significativa del loro peso basale dopo sfida con C. difficile R20291. Il peso corporeo medio di topi è stata misurata ogni giorno dalle giorno 0. Significativa perdita di peso è stato visto nei giorni 2 a 7 rispetto al giorno 0, il peso della linea di base. C) C. difficile R20291 carica batterica nelle feci dopo la sfida. Entro 24 ore dopo la sfida con spore di C. difficile, tutti i topi sono stati colonizzati con 10 7 CFU (unità formanti colonia) per g di feci. Non ci sono differenze significative in questi parametri sono stati osservati tra femmine e maschi. La significatività è stata determinata con il test ANOVA non parametrico di Kruskal-Wallis a senso unico seguito da post-test di Dunn (*, p ≤ 0,05; **, p ≤ 0,01; ***, p ≤ 0,001; ****, p </em&gt; ≤ 0,0001). Le barre di errore rappresentano le deviazioni standard dalla media. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Quattro topi (2 maschi e 2 femmine) sono stati umanamente eutanasia e preparati per la necroscopia al giorno 0, prima di infezione, per servire come controlli non infetti. Inoltre, un mouse da ciascuna gabbia era umanamente eutanasia e preparato per necroscopia nei giorni 2, 4, 7, e 14 post-sfida (Figura 1A). Conteggio di C. difficile all&#39;interno del contenuto del cieco è stato eseguito ad ogni necroscopia. In accordo con l&#39;enumerazione fecale, nessun C. difficile è stato rilevato nel contenuto cecale di topi prima della sfida con C. difficile. A 48 ore post-sfida, i topi sono stati colonizzati con circa 10 CFU 8 di C. difficile per g di contenuti del cieco (Figura2A). Tutti i topi sono rimasti persistentemente colonizzato tutto l&#39;esperimento. Contenuto del cieco è stato valutato anche durante l&#39;infezione per la presenza di tossina di C. difficile utilizzando il test di citotossicità su cellule Vero. Nessuna evidenza di C. difficile tossina citotossicità è stata rilevata nel contenuto cecale precedenti alla sfida (Figura 2B). C. citotossicità difficile è stato rilevato 2 giorni dopo la sfida con spore di C. difficile e persisteva per tutta l&#39;infezione. Nonostante la colonizzazione abbastanza uniforme con C. difficile, variazione dei livelli di C. difficile attività citotossica è evidente in topi individuali (Figura 2B). La maggior parte dei contenuti del mouse cecal neutralizzato con antitossina di C. difficile durante il test di citotossicità delle cellule Vero e non aveva alcun segno di tossicità (Vero cell arrotondamento) nelle diluizioni effettuate. Tuttavia, alcuni topi individuali, nonostante retesting, avevano evidenza di 50 - 100% citotossicità in 10 -1 di diluizione (diluizione dove il campione è più concentrata), senza evidenza di cellule Vero arrotondamento nelle altre diluizioni. L&#39;antitossina utilizzato in questo saggio è un policlonale anticorpi neutralizzanti al C. difficile tossina, preparata nelle capre 27. Pertanto, questo antitossina non può neutralizzare la tossina di C. difficile binario che viene prodotta dal ceppo R20291. Tuttavia, la tossina binaria C. difficile non è considerato essere citotossici 10. Un&#39;eccessiva C. difficile tossina in grado di essere neutralizzato l&#39;antitossina o la presenza di un altro agente citotossico diverso C. difficile tossina potrebbe contribuire a questo risultato. Questa osservazione non influenzare la determinazione del titolo citotossicità per questi campioni, dallo scorso la diluizione di ogni campione con l&#39;arrotondamento delle cellule Vero 80% ha avutonessun segno di tossicità quando combinato con l&#39;antitossina alla diluizione specificata. <img alt="figura 2" src="/files/ftp_upload/54850/54850fig2.jpg" /> Figura 2: La colonizzazione del cieco e citotossicità durante l&#39;infezione con C. difficile R20291. A) Mouse ceca è rimasto persistentemente colonizzato da C. difficile tutto l&#39;esperimento, nonostante la risoluzione dei segni clinici e l&#39;aumento di peso osservato. Tutti i topi sono stati colonizzati con più del 10 8 UFC per g di contenuto cecale quando valutati a 2 giorni dopo la sfida. B) test di citotossicità delle cellule Vero dal contenuto del cieco durante l&#39;infezione da C. difficile R20291. Dopo la sfida con spore di C. difficile, i topi avevano livelli significativi di citotossicità, misurata in log 10 di diluizione reciproco di tossina per g di contenuti del cieco nei giorni 2, 4, e 7 post-sfida rispetto al giorno 0 (mediane represented dalla linea). La significatività è stata determinata con il test ANOVA non parametrico di Kruskal-Wallis a senso unico seguito da post-test di Dunn (*, p ≤ 0,05; **, p ≤ 0,01; ***, p ≤ 0,001; ****, p ≤ 0,0001 ). Le barre di errore rappresentano le deviazioni standard dalla media. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Si raccomanda che la valutazione istologica è condotta da un patologo veterinario bordo certificata in modo cieco. Per il punteggio del tessuto, un sistema di punteggio numerico 0-4 dovrebbe essere impiegato per valutare separatamente l&#39;edema, infiltrazione di cellule infiammatorie, e danni alle cellule epiteliali nel ileo, cieco, colon e sulla base di uno schema di punteggio precedentemente pubblicato 17. Su esame istopatologico accecatoinazione del murino ileo, cieco, e due punti che seguono sfida con C. difficile R20291, le più significative alterazioni patologiche sono stati notati nel cieco (Figura 3). I punteggi totali istologiche cecale erano significativamente differenti tra il giorno 0 e giorni 2 e 4 post-sfida. Nessuna lesione significativa è stata notata nell&#39;ileo tutta infezione (Figura 3). Le lesioni più lievi sono stati notati nel colon. I punteggi totali istologici del colon sono risultati significativamente differenti tra il giorno 0 e giorni 2 e 7 Post sfida (Figura 3). Rappresentante H &amp; E sezioni del ileo, cieco e del colon in tutto infezione sono disponibili nella figura 4. Ogni immagine ha il suo rispettivo punteggio istologico totale e punteggi singoli per danno epiteliale, infiammazione ed edema, come determinato da un patologo veterinario bordo certificata cieco ( SAM). Figura 3: punteggio istologico del Murine Ileo, Intestino cieco, e del colon durante l&#39;infezione con C. difficile R20291. Totale punteggi istologici sono stati calcolati sommando tutti e tre i punteggi dei parametri valutati: epiteliale danni, infiammazione ed edema. Non sono significativi cambiamenti istopatologici sono stati notati nell&#39;ileo tutta infezione. tessuto cecal conteneva le lesioni istologiche più significativi durante CDI. I punteggi totali istologici cecale erano significativamente differenti dal giorno 0 nei giorni 2 e 4 post-sfida. Le lesioni più lievi sono stati notati nel colon. I punteggi totali istologici del colon erano significativamente differenti dal giorno 0 nei giorni 2 e 7 post-sfida. La significatività è stata determinata con il test ANOVA non parametrico di Kruskal-Wallis a senso unico seguito da post-test di Dunn (*, p ≤ 0,05; **, p ≤ 0,01; ***, p804; 0.001; ****, P ≤ 0,0001). Le barre di errore rappresentano le deviazioni standard dalla media. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54850/54850fig4.jpg" /> Figura 4: istopatologia durante l&#39;infezione con C. difficile R20291. Questo pannello contiene rappresentativi sezioni H &amp; E di ileo, cieco e del colon in tutto infezione da C. difficile R20291. I punteggi istologici totali sono tra parentesi () per il corrispondente giorno e tessuti elencati: per il giorno ileo 0 (0), il giorno 2 (1), il giorno 4 (0), il giorno 7 (0), e il giorno 14 (0) ; per il giorno cieco 0 (0), giorno 2 (5), il giorno 4 (4), il giorno 7 (3), e il giorno 14 (3); e per il giorno colon 0 (0), giorno 2 (4), giorno 4 (1), il giorno 7 (4), e il giorno 14 (2). Microfotografie sono stati ottenuti su un Microscopio di lucee con una fotocamera digitale da 5 megapixel e il relativo software (la barra della scala rappresenta 200 micron). <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54850/54850fig4large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291 at JoVE.com

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Questo protocollo delinea il modello di topo cefoperazone di infezione da Clostridium difficile (CDI) con un ceppo clinicamente rilevante e geneticamente trattabili, R20291. L&#39;enfasi sul monitoraggio clinico della malattia, C. enumerazione batterica difficile, la tossina citotossicità, e cambiamenti istopatologici tutto CDI in un modello di topo sono dettagliati nel protocollo.

Cefoperazone trattati modello murino di clinicamente rilevanti<em&gt; Clostridium difficile</em&gt; R20291 Strain

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

cefoperazone-treated-mouse-model-clinically-relevant-clostridium

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

12.2K Views.  North Carolina State University College of Veterinary Medicine.  Questo protocollo delinea il modello di topo cefoperazone di infezione da Clostridium difficile (CDI) con un ceppo clinicamente rilevante e geneticamente trattabili, R20291. L'enfasi sul monitoraggio clinico della malattia, C. enumerazione batterica difficile, la tossina citotossicità, e cambiamenti istopatologici tutto CDI in un modello di topo sono dettagliati nel protocollo. 

Video: Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Coltura e manutenzione<em&gt; Clostridium difficile</em&gt; In un ambiente anaerobico

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Ricerca

Immunologia e infezioni

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

La prevenzione di stress termico effetti avversi nei ratti<em&gt; Bacillus subtilis</em&gt; Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

Un protocollo per caratterizzare i cambiamenti morfologici di<em&gt; Clostridium difficile</em&gt; In risposta al trattamento antibiotico

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Studi del Virus di riossidazione A in un modello murino di infezione

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Replica di ordinato, Nonredundant biblioteca del ceppo di Pseudomonas aeruginosa PA14 trasposone inserimento mutanti

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

Un'analisi di Microarray della proteina per determinazione sierologica dell'infezione di Clostridium difficile anticorpo seguenti risposte antigene-specifiche

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Una purificazione e l'analisi di attività In Vitro per un ppGpp (p) sintetasi da Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Un modello di Mouse controllata per sepsi polimicrobica neonatale

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

Misurazione della velocità di propagazione dell'impulso, distensiabilità e deformazione in un modello di topo abdominato di aneurisma aortico

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

Costruzione di mutanti nel sierotipo 1 Streptococcus pneumoniae ceppo 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...