Observatorio de Comercio Exterior Informe de omercio Actualización Exterior Intercambio con Principales Socios Departamento de Economía Gabriel Molteni Gonzalo De León Martín Mattiazzi Panorama General En el mes de febrero de 2014 las exportaciones argentinas fueron de 5393 millones de dólares, lo que representó un crecimiento de 3,1% en relación al mes anterior. En la comparación interanual se detectó una caída de 6,1%, explicada por un descenso en las cantidades vendidas de 5% y una disminución de los precios de 1 por ciento.
Beneficial microbesBeneficial Microbes, March 2013; 4(1): 39-51 Wageningen Academic P u b l i s h e r s Probiotics in Clostridium difficile infection: reviewing the need for a multistrain
M. Hell1,2, C. Bernhofer1, P. Stalzer1,2, J.M. Kern2 and E. Claassen3,4
1Department of Hospital Epidemiology and Infection Control, Salzburg University Hospital, Paracelsus Medical University, Strubergasse 21, 5020 Salzburg, Austria; 2Division of Medical Microbiology, Institute of Laboratory Medicine, Salzburg University Hospital, Paracelsus Medical University, Strubergasse 21, 5020 Salzburg, Austria, 3Erasmus Medical Centre, Department of Virology, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands; 4Athena Institute, VU Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands; Received: 24 May 2012/ Accepted: 4 February 2013 2013 Wageningen Academic Publishers In the past two years an enormous amount of molecular, genetic, metabolomic and mechanistic data on the host- bacterium interaction, a healthy gut microbiota and a possible role for probiotics in Clostridium difficile infection (CDI) has been accumulated. Also, new hypervirulent strains of C. difficile have emerged. Yet, clinical trials in CDI have been less promising than in antibiotic associated diarrhoea in general, with more meta-analysis than primary papers on CDI-clinical-trials. The fact that C. difficile is a spore former, producing at least three different toxins has not yet been incorporated in the rational design of probiotics for (recurrent) CDI. Here we postulate that the plethora of effects of C. difficile and the vast amount of data on the role of commensal gut residents and probiotics point towards a multistrain mixture of probiotics to reduce CDI, but also to limit (nosocomial) transmission and/or endogenous reinfection. On the basis of a retrospective chart review of a series of ten CDI patients where recurrence was expected, all patients on adjunctive probiotic therapy with multistrain cocktail (Ecologic®AAD/OMNiBiOTiC® 10) showed complete clinical resolution. This result, and recent success in faecal transplants in CDI treatment, are supportive for the rational design of multistrain probiotics for CDI.
Keywords: Clostridium difficile, colonisation of GI tract, colonisation resistance, diarrhoea, intestinal mucosa
C. difficile is a gram-positive, anaerobic, spore forming bacillus first described in 1935 after isolation from the stool Clostridium difficile infection (CDI) is the most significant of a healthy newborn (Bacillus difficilis; Hall and O'Toole, bacterial cause of hospital acquired (nosocomial) diarrhoea 1935). Normally, neonates develop a stable microbiota (even in adults (Bauer and Van Dissel, 2009). The severity of when colonised with C. difficile) without clinical problems, CDI ranges from mild, usually self-limiting, diarrhoea to probably because they are short of (adequately expressed) fulminant colitis, toxic megacolon and death. C difficile toxin receptors in the still immature gut. Not until 1978 colonises and can be isolated from 0-5% of healthy adults was C. difficile recognised as an opportunistic pathogen for (Hautmann et al., 2011; Hell et al., 2012; Moudgal and its antibiotic-associated diarrhoea and pseudomembranous Sobel 2012; Parkes et al., 2009). However, this can increase colitis. CDI has rapidly increased since the 1990s with to 39% in hospitalised patients (Hickson, 2011; McFarland, alarming rise since 2000 (1999-2007: 25% incidence- 2011) depending on treatment and local conditions, increase per year in USA and 750% fatality rate increase with individuals over the age of 65 being prime targets in UK; Kelly, 2009) when the novel and fluoroquinolone and age itself being a predisposing factor (referred to as resistant strain of PCR ribotype 027 spread and accounted ‘inflammaging' and ‘immunosenescence'; Islam et al., 2012). for severe disease and over 40% of isolates (Islam et al., ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2012.0049 39 M. Hell et al.
2012). Currently, CDI incidence rates are still high, but and are non-invasive, they will disappear (average half life have dropped due to rapid diagnosis, improved infection 5-7 days) when no longer ingested (Mercenier et al., 2000).
control practice, root cause analysis, isolation of cases/ patients, and restricted use of antibiotics (stewardship). Clostridum difficile specific aspects
Since the problem with nosocomial infections also lies with spore-contaminated facilities and asymptomatic carriers CDI has a number of specific properties making it the (high IgG anti-toxin titers; Kyne et al., 2000), transmission major nosocomial diarrhoea in adults. Firstly, CDI is reduction, in patients and health-care-workers alike, is an particularly known for its specific risk factors age, use essential element in infection control.
of antibiotics and hospitalisation (Hickson, 2011). When properly diagnosed, CDI is usually treated by withdrawal 2. Clostridium difficile infection (mainly
of the precipitating antibiotic, avoidance of anti-peristaltic clinically manifested as diarrhoea)
agents and treatment with metronidazole or vancomycin for non-metronidazole-responders (Cohen et al., 2010) or severe cases. Still, up to a quarter of all patients will develop recurrent CDI (Hickson 2011), with those that experience The normal microbiota inhibits (opportunistic) pathogen such a relapse having a 50-60% risk of further recurrence growth and toxin release. This function is reduced after (Bauer and Van Dissel, 2009). This is particularly so because gut-damage and demonstrably so in faecal samples of of hypervirulent strains (like ribotypes 027, 078 and 106) antibiotic-treated patients (Parkes et al., 2009). Causes with more severe disease, increased mortality, resistance for microbiota-damage are shown at level 2 in Figure 1. to fluoroquinolones and higher relapse rates (Cartman Risk factors for microbiota damage are for example age, et al., 2010). Additional mortality by these hypervirulent proton pump inhibitors (PPI), tube feeding and parenteral strains is calculated to be between 6-12% (Parkes et al., nutrition (Hautmann et al., 2011). After initial disturbance 2009). Although some of these recurrent cases are due to of the resident microbiota other factors can exacerbate the exogenous reinfection by ongoing exposure to spores in condition by causing an unwanted bacterial overgrowth the environment, most exhibit the same bacterial strain (Figure 1: 3) resulting in a mild (osmotic) diarrhoea of the first episode. Evidently, neither the first antibiotic that is usually self limiting (in over 75% of cases). When nor vancomycin restored the gut microbiota, nor did they bacterial toxins are produced in sufficient amounts they reduce the exposure to spores in the environment, the co- will bind and structurally damage epithelial cells and/or morbidity or other CDI specific host risk factors (Bauer the tight junctions, and the gut barrier is compromised and van Dissel, 2009). Fidaxomicin, a recently available (frequently also leading to bloody stools). This will then anti-CDI drug has a very specific mode of action against C. lead to an inflammatory cascade also involving the nervous difficile and does, therefore, less harm to the gut microbiota system, which intensifies the diarrhoea. The reduction in than any other comparable anti-C. difficile antibiotics fermentation also gives a reduction in the production of (Tschudin-Sutter et al., 2012). Fidaxomicin is also described short chain fatty acids (SCFA) which normally provide as preserving the intestinal microbiome during and after energy and stimulation to colonocytes. Again, a large part of treatment of CDI and reducing both toxin reexpressions these violent episodes of diarrhoea is self limiting. When the and recurrence of CDI (Louie et al., 2012).
immune system, diet and general condition of the patient are optimal, the resident microbiota will return and the cellular CDI starts with ingested (or resident) spores germinating damage will be repaired by normal cellular regeneration in the colon and the bacteria establishing/maintaining (from crypt to apex of the villi 3-5 days). Prebiotic fibres themselves through specific adhesion (Islam et al., 2012). like galacto- and fructo-oligosaccharides will accelerate CDI is toxin mediated (Figure 1: 4). Two large (approx. restoration by preferentially creating a beneficial bifidogenic 300 kDa) protein exotoxins, TcdA and TcdB, are produced, milieu whereas ‘simple' sugars will enhance growth of of which TcdB is clearly the main virulence factor as opportunistic pathogens (Kelly, 2008). Similar beneficial demonstrated by TcdA-veB+ve strains. The hypervirulent effects as with prebiotics (specific vegetables in diet and/ strain ribotype 027 produces an additional ‘binary' toxin or supplements like inulin) can be observed by oral intake (CDT, an actin-specific ADP ribosyltransferase) whose of billons probiotic bacteria). Since the bacteria in the gut role is not yet established, although it potentiates TcdA/B will always be counteracted by the (healthy) immune system toxicity (Tschudin-Sutter et al., 2012). TcdA binds to the (gut associated lymphoid tissue) only those bacteria that apical side of gut epithelial cells (to gp96, a C. difficile toxin were considered ‘self' in early life will persist in view of the A receptor) and causes cytoskeletal modification and tight gut immune tolerance they enjoy. Hence, the same profile junction disruption. This in turns facilitates binding of fingerprint of bacteria will emerge after a dysbiosis episode. TcdB to the basal lamina leading to vascular permeability, In any case, the balance is usually restored after several release of neuropeptides (substance P, CGRP/calcitonin weeks. Since probiotics do not permanently colonise the gut gene-related peptide and neurotensin), recruitment of white blood cells, pro-inflammatory cytokines (leukotrienes, Beneficial Microbes 4(1) Probiotics in Clostridium difficile infection 1. Homeostasis of gut microbiota shown as normal distribution of beneficial (green) and unwanted (red) commensals and/or intruders 2. Damage to microbiota through e.g.: – Antibiotics or chemotherapy – Diet or toxic compounds – Trauma or stress (inc. colonoscopy, surgery) – Anti-microbiota auto-immune response (e.g. IBD) 3. Overgrowth of potentially pathogenic bacteria – Faulty diet or malnutrition – Deficient immune response – Fermentation ↓ → SCFA ↓ & carbohydrates ↑ → osmotic pressure ↑ → colon absorbs less → liquid stools 4. Damage to cells and barrier (can also be step 2) – Toxins (red dots) give cytoskeletal modification – Disruption of tight junctions loss of barrier – Loss of absorptive microvilli (effacement) – Epithelial detachment (pseudomembranes) 5. Leaky gut barrier and infiltration – Cytotoxins bind to basolateral membrane leading to pro-inflammatory cytokines, vascular permeability, damage to connective tissue, infiltration of white blood cells, epithelial cell apoptosis. Release of neuropeptides stimulate enteric nerves leading to fluid loss: diarrhoea 6. Beneficial bacteria return in numbers – Consumption of probiotic (purple) – Dietary changes (e.g. prebiotics) – Immune system (GALT) – Time (self limiting diarrhoea) 7. As 1. Balance restored, bacterial profile (faecal fingerprint) as before due to memory immune response (probiotics don't colonise & will disappear) Figure 1. Homeostasis, damage, barrier/function-loss and repair of the gut epithelium: role of microbiota and probiotics.
SCFA = short chain fatty acids; GALT = gut-associated lymphoid tissue.
3. Probiotics for prevention and treatment of
2, TNFα, IL-1, IL-6) epithelial cell apoptosis, pseudomembrane (PM-colitis) formation, connective tissue degradation, fluid exudation/secretion and subsequently diarrhoea and frequently also bloody stools. In short, C Maintenance of homeostasis and luminal effects
difficile is pathogenetically unique in establishing a bona fide necro-inflammatory reaction activating mast cells, As shown in Figure 2, both the commensals in a normal nerves, vascular endothelium and immune cells in addition microbiota as wel as ingested probiotic products will assist to disruption of tight junctions (Hodges and Gill, 2010).
in defending the gut against colonisation by exogenous microorganisms. This mechanism is called colonisation resistance (Wolvers et al., 2010). In a single cell to single Beneficial Microbes 4(1) M. Hell et al.
Table 1. Contributions of electrogenic versus electroneutral components of ion absorption at the basis of Clostridium difficile
Diarrhoea because of Mechanism through
Molecules involved Relevant for C. difficile
Role of probiotics c
Increased secretion of Overproduction of cAMP Increased Cl- secretion through Bifidobacteria dose dependently electrolytes (CLCA) 1 gives activation of protein neuropeptides (substance P, inhibit (CFTR) Cl- secretion3 and kinase A and opens CFTR CGRP and neurotensin) promote intestinal homeostasis and Cl- secretion2 one step downstream epithelial Ca2+ mobilisation Luminal membrane Na+ and NHE3 (aka SLC9A3) TcdB inactivates Rho-kinase Butyrate produced by probiotics H+ exchange isoforms1,3 inhibitor altering activity and increases NaCl absorption distribution of NHE34 by NHE3 stimulation and transcription5 Reduced chloride Apical anion exchange As for other infectious diarrhoea LPA or Lactobacillus acidophilus mediating Cl- absorption increase surface expression of (seen in congenital DRA giving increased chloride chloride diarrhoea) ENS links directly with AQP8 AQP, ANG, OXT Hypothesis: lactic acid upregulates9 Consumption of L acidophilus AQP4, which facilitates oedema led to higher gene expression elimination in diseases causing of water and ion homeostasis vasogenic10 (vessel leak) Reduced sodium and Inactivation of SGLT-1 As for other infectious diarrhoea12 Microbiota influences expression glucose absorption Altered tight junctions14,15 claudin-1, ZO-1, TcdA modifies cytoskeleton and Probiotics upregulate genes function, increased disrupts tight junctions coding for de novo synthesis of claudin and occludin16,17 a Abbreviations used: ANG = angiogenin; AQP = aquaporins; CFTR = cystic fibrosis transmembrane conductance regulator; CLCA = chloride channel accessory; DRA = down regulated in adenoma; ENS = enteric nervous system; LPA = lysophosphatidic acid; NHE3 = Na/H exchanger3; OXT =oxytocin; SGLT-1 = sodium D-glucose cotransporter; TcdA = toxin A; ZO = zonula occludens (zonulin).
b Specific references given as superscript letters: 1Hodges and Gill, 2010; 2Ohland et al., 2012; 3Heuvelin et al., 2010; 4Hayashi et al., 2004; 5Malakooti et al., 2011; 6Borthakur et al., 2008; 7Singla et al., 2011; 8Ishihara et al., 2008; 9Morishima et al., 2008; 10Saadoun and Papadopoulos., 2010; 11Van Baarlen et al., 2011; 12Dean et al., 2006; 13Swartz et al., 2012; 14Ulluwishewa et al., 2011; 15Veshnyakova et al., 2012; 16Karczewski et al., 2010; 17Wells, 2011.
c Strains as examples, specifics in references/primary papers.
cell communication mechanism called quorum sensing of virulence factors, e.g. adherence molecules normally some bacteria are able to down-regulate gene expression expressed by pathogens (Cadieux et al., 2009). Not only of pathogens, thereby decreasing virulence factors and/ does the production of these compounds prevent dysbiosis or growth (Sherman et al., 2009). Furthermore, lactic acid in the gut, but this also explains why lactic acid bacteria bacteria produce a plethora of anti-microbial compounds have been used successfully for over three thousand years to including SCFAs, such as acetic, propionic, caproic conserve and sensorically improve food (milk, beer, sausage, and butyric acid, but also hydrogen peroxide, ethanol, sauerkraut, pickles, cheese, wine, etc.). Furthermore, SCFAs, acetaldehyde, diactyl, and carbon dioxide – all derived as like butyric acid, can also double in function as colonocyte either oxygen-catabolites or sugar-catabolites. Similarly, fuel and can stimulate gut motility. Needless to say that toxic compounds as fat and amino acid metabolites are efficient probiotics should share at least some of these, produced, such as 3- and 4-hydroxy fatty acids, phenyllactic largely metabolomic, characteristics to be effective in vivo.
acid, aromatic and heterocyclic molecules. De novo protein synthesis by lactic acid bacteria results in antifungals, bacteriocins like reuterin and reutericyclin, and a host of low molecular mass peptides and cyclic peptides (see De Vuyst Antimicrobial factors are not only made by luminal lactic et al., 2009). Next to the bactericidal and bacteriostatic acid bacteria but also produced by the Paneth cells and actions, these compounds can also downregulate expression secreted in the lumen at the mucosal surface, aiding in host Beneficial Microbes 4(1) Probiotics in Clostridium difficile infection Exclusion by competition for: – Nutrients – Space in lumen & staeric hindrance ( → in figure) – Adherence to gut epithelium – Quorum sensing effector mechanisms Bacterial metabolic activity (green asterisks) – Acids ↓ pH & boost beneficial commensals– Production of bactericides and bacteriostats– Release of gut stimulators (e.g. butyrate) – Proteases for hydrolysis of toxins Preservation of gut-barrier function – Upregulate synthesis tight junction proteins ( ↑↑ in figure)– Decrease macromolecular permeability – Reduce bacterial translocation Influence water and ion channels – Probiotics inhibit secretion, stimulate absorption and upregulate gene expression and transcription of essential transport molecules Influence nervous system – Enteroendocrine cells and subepithelial nerves Modulation of signal transduction – Block activation & translocation of IFN-γ and transcription factors like NF-κB to nucleus – Idem for mitogen-activated protein kinases (MAPK)– Enhance synthesis anti-inflammatory cytokines like IL-10 and TGF-β Stimulate innate immune system – Mucus production (Goblet cells), lysozyme – MAMPS microbe associated molecular patterns – Modulate TLR's, RLR's and NLR's – Trigger Paneth cells to produce alpha-defensins Initiate and boost adaptive immunity – Antigen uptake/presenting M & dendritic cells – From Peyer's patches to mesenterical lymph nodes – Immune-cytokines (Th and Tregs) – IgA, IgG and Ig antibodies – Recirculating memory cells and HEV's Figure 2. Microbiota, gut mechanisms and cells involved in prevention or reversal of diarrhoea.
IFN = interferon; NFκβ = nuclear factor kappa beta; IL = interleukin; TGF = transforming growth factor; MAMPS = microbe-associated
molecular patterns; TRL = Toll-like receptor; RLR = retinoic acid inducible gene 1 like receptor; NRL = nucleotide oligomerisation
domain like receptor; IG = immunoglobulin; HEV = high endothelial venules.
defence by affecting numbers and/or composition of the expression can predispose for pathology, seen in necrotising colonising microbiota. As an integral part of the immediate enterocolitis and ileal Crohn's disease (Salzman et al., 2007).
response innate immune system, Paneth cells produce defensins and other antibiotic peptides and proteins. Probiotics can enhance release of defensins, e.g. as Because of their juxtaposition to epithelial progenitor (stem) demonstrated in acute infectious enteritis (see Sherman cells at the base of the crypts of Lieberkűhn and very high et al., 2009). Trefoil-factors are anti-bacterial peptides that local concentrations of defensins, they are probably involved are also secreted by mucin-producing cells in response in maintaining gut renewal. Reduced Paneth cell defensin to various noxious stimuli (Sherman et al., 2009) or Beneficial Microbes 4(1) M. Hell et al.
probiotics (Van Huynegem et al., 2009). The enhanced M (microfold) cells, exclusively located over the Peyer's mucus layer overlying the epithelial lining of the gut serves patches, in the gut epithelium continuously sample the as an additional antibacterial shield, hampering binding lumen for particles like microorganisms, transferring of the pathogens (Sherman et al., 2009). Probiotics, such antigens to dendritic cells in the submucosa. Intraepithelial as Lactobacillus plantarum, can upregulate MUC2 and T and B lymphocytes produce cytokines (T) and MUC3 genes, which code for mucus-protein production immunoglobulins (B; antibodies) of IgA isotype mainly in humans. Mucus and trefoil factors work in concert with (Bron et al., 2012). Finally, the gut barrier, formed by only each other and many of the more than twelve hundred a single layer of epithelial cells, is critically dependent on (!) antimicrobial peptides nature can call upon for host tight junctions separating the gut lumen from the lamina defence (Nakatsuji and Gallo, 2012). The gut wall exhibits propria. Bacteria from the microbiota and probiotics alike a large degree of luminal chemo-sensitivity sensing a target the tight junction proteins and thereby modulate the vast array of signals ranging from nutrients, chemicals, barrier and thus permeability (Ulluwishewa et al., 2011).
mechanical factors and microorganisms (Nguyen, 2012; Raybould, 2012). Entero-endocrine cells are specialised luminal sensors as are sub-epithelial nerve fibres that will respond to those compounds freely diffusing across the The ‘business end' of the gut immune system is mainly epithelium (e.g. SCFAs; Dockray, 2003).
present in the lumen as sIgA immunoglobulin in the mucus layer on top of the epithelial cells and as intraepithelial Another direct relation between the nervous system and the lymphocytes. However, the actual initiation of the gut is the presence, and exquisite sensitivity to probiotics, of immune response takes place in the Peyers's patches, large opioid and cannabinoid receptors enabling manipulation of lymphocyte follicles in the submucosa. After the antigens visceral perception (including pain; Rousseaux et al., 2007). are presented (by dendritic cells in villi or through M cel s) As described in detail in Table 1, an intimate association and T and B cells are recruited the actual immune response between sensing, the nervous system, and ion and water is started in the draining mesenterical lymph nodes from homeostasis exist (Hodges and Gill, 2010). Sensing is also an where the activated T and B cells are transported back to the integral part of the immune system via pattern recognition villi via the blood vessels (Brandtzaeg and Pabst, 2004; Bron receptors (PRR) expressed on immune cells like dendritic et al., 2012). This seemingly elaborate mechanism ensures cells and other tissues like the gut epithelium. PRRs that the villi have more room for their primary function, recognise evolutionary conserved molecular structures uptake of nutrients and water, and that specific immune known as microbe-associated molecular patterns or effector-cells can be initiated at one single site (after pathogen-associated molecular patterns and signal effector pathogen recognition) and then be evenly redistributed mechanisms in the innate immune system (Olive, 2012). over the entire length of the gut, thereby providing uniform PRRs can be divided in three families: Toll-like receptors, specific protection and memory. The entire process is retinoic acid inducible gene 1 like receptors recognising critically controlled by dendritic cells and effector-cells viruses, and nucleotide oligomerisation domain like are always under control of T regulator cells, ensuring receptors. All are three families amenable to modulation not only a decent start of the specific response but also, by probiotics (Feleszko et al., 2006), excellently reviewed and maybe more importantly, a timely end to the response and described in their innate and adaptive immunity context (Wel s, 2011) avoiding self-inflicted pathology (Chinen and by Wells (2011).
Rudensky, 2012; Van Driel and Ang, 2008).
Based on these and other sensing mechanisms, relevant Clinical use of probiotics
signal transduction pathways (see Figure 2 for NFkappaB and mitogen-activated protein kinase) can be switched With over 700 clinical trials in healthy volunteers and on after recognition of pathogens leading to a cascade patients one might conclude that probiotics have come of events ending in the production of proinflammatory of age and their use is as evident as the mechanisms they cytokines (as described above, cytokine storm) for defence modulate such as described above. Unfortunately, effects or anti-inflammatory cytokines when tolerance is needed of probiotics are always dose and strain specific making (Bron et al., 2011; Hodges and Gill., 2010; Sherman et al., comparison of clinical results with different preparations 2009; Van Baarlen et al., 2009). Selected probiotic strains very difficult. In an effort to at least indicate in which areas and environmental markers of microbial exposure (Ege probiotics can be recommended on scientific grounds, et al., 2011) can selectively modulate these pathways and Floch et al. (2011) made an update of existing data. In enhance or suppress cytokine production switching the their analysis they listed evidence as: ‘A = strong positive immune system between better defence (pathogens, tumour studies in literature', ‘B = positive-controlled studies, cells) and/or tolerance (to avoid allergy and auto-immunity; but some negative studies not supporting the primary Guarner et al., 2006;).
outcome' and ‘C = some positive studies, but not enough for certainty'. They concluded that A claims can only be Beneficial Microbes 4(1) Probiotics in Clostridium difficile infection given for infectious diarrhoea in children, antibiotic- associated diarrhoea (AAD), pouchitis, ulcerative colitis (maintenance), immune response and atopic eczema. In A patient was identified as a laboratory confirmed, most of these clinical indications a combination of probiotic symptomatic CDI patient who received adjunctive probiotic strains instead of a monospecific single strain was used therapy at the time when oral metronidazol or vancomycin (Chapman et al., 2011).
was initiated. Participants consumed sachets containing 5 g Ecologic®AAD twice daily. Patients were studied by queering Clinical use of probiotics in Clostridium difficile infection
surveillance data, consecutively followed by interviewing staff by phone. Retrospectively, medical records were In the studies by Floch et al. (2011), prevention of (recurrent) reviewed to ascertain clinical signs of CDI, therapy, medical C. difficile associated diarrhoea was given a disappointing history and outcome.
B/C marking and only one bacterial product with one strain (Lactobacillus rhamnosus GG; ATCC 53103) was Severe CDI was defined by clinical signs of severe colitis and included. Consequently, even with a substantial number of laboratory findings confirming a severe course; recurrent studies (Malaguarnera et al., 2012) and very positive meta- CDI was defined as described by Bauer et al. (2009). analysis showing probiotics are associated with a reduction The decision to initiate adjunctive probiotic therapy was in AAD (Hempel et al., 2012; Videlock and Cremoni, 2012), a made by the individual attending physician in cooperation reliable probiotic formulation for CDI has still to be clinically with an infectious diseases consultant. On the basis of (significance in intent-to-treat) proven. Still a positive retrospective chart review all patients met the following attitude towards the future success of anti-CDI probiotics criteria: diarrhoea, antibiotic treatment with metronidazole can be seen and specific recommendations for use are given or vancomycin and multistrain probiotics.
(Hickson, 2011). Single strain versus multistrain preparations are still discussed controversially (Chapman et al., 2011). Clinical setting and inclusion criteria
There is some evidence for single strain applications, such as non-toxigenic C. difficile strains, for treatment (Phase 2 A 1,200 bed, tertiary care, university hospital (five different studies) based on the idea of a monoclonal pathogenesis clinical departments) participated during the period from of toxigenic C. difficile (Hell et al.2011). But a number of 1 November 2010 to 31 July 2011. After laboratory proof of factors have to be taken into account when developing a C. difficile (toxigenic culture), a standardised interview was new probiotic formulation in this field, these being: age of carried out for each patient, asking for date and reason of the host, optimal dose, stability, safety, mucosal adherence, admission, clinical symptoms, onset of clinical symptoms, gastric acid and bile resistance, matrix of delivery, specific underlying diseases, comorbities and antibiotic history. If strains, interaction within combination products and the patient was selected for the study, patients' records optimal duration of treatment (Verna and Lucak, 2010). were searched for antibiotic history, additional medication The high efficiency of faecal microbiota transplantation is like PPIs and cortison, fever, leukocyte counts, C-reactive a fundamental fact that can be relied on in probiotic CDI protein (CRP), radiology and endoscopy results. A follow- treatment (Brandt., 2012; Tschuddin-Sutter et al., 2012). up was done after six month.
Based on this, a recent metanalysis by Johnston et al., 2012) and the fact that C. difficile has unique necro-inflammatory Treatment description and definition of resolution
pathogenesis, as described in detail in Section 2, we postulate that only a multistrain cocktail (resembling a ‘healthy' human To test whether the product was actually performing microbiota) could come close to addressing all mechanistical and no strain to strain inhibitory effects were introduced needs (Figure 2 and Table 1) in the CDI setting.
by combining so many strains, a pilot study including a series of ten elderly patients, complemented by a detailed 4. Design and methods of a clinical study
chart review, was performed. Resolution was defined as no further laboratory signs of inflammation and/or fever, For the reasons listed in the previous section a product and reduction of stool frequency less than 3 times/24 h for (Ecologic®AAD, Winclove Bio Industries BV, Amsterdam, at least 72 h according to Bauer et al. (2009).
the Netherlands) was assembled consisting of equal ratios of the following 10 bacterial strains with a total dose of 5 All patients received oral vancomycin 125 mg qid for at least g/sachet and of 109 cfu/g: Bifidobacterium bifidum W23, 10 days, 8 patients received additionally oral metronidazole Bifidobacterium lactis W18, Bifidobacterium longum W51, 500 mg tid, 4 patients got a combination of oral vancomycin Enterococcus faecium W 54, Lactobacil us acidophilus W37 and iv treatment with metronidazole, and 9 were stil under and W55, Lactobacil us paracasei W20, L. plantarum W62, concomitant iv treatment with other antibiotics because L. rhamnosus W71, Lactobacillus salivarius W24 and a of their underlying disease.
mixture of 5% mineral elements, in Austria branded as OMNiBiOTiC® 10 AAD)).
Beneficial Microbes 4(1) M. Hell et al.
A complementary epidemiological investigation was done Laboratory and radiography results
outside the hospital CDI surveillance system for both the period of the investigated cases and for the time period In all patients, CDI had been confirmed by laboratory 2009-2011 (Table 2) to gain more background information results (stool culture for C. difficile and toxin A- and of the local/hospital epidemiological situation.
B-positive ELISA test) before they were included in the survey. Radiography and endoscopy brought no further 5. Results of a clinical study
useful information: in 3 cases abdominal X-rays were done but without description of the colon; in one case an Table 2 clearly shows that the caseload of CDI is abdominal CT scan was performed, but, as the patient was proportionally much higher in the group under 70 years already known for chronic radiogenic colitis, there was no of age, with 521 cases under 70 years of age versus 452 in valid information about CDI. In 2 out of 10 cases endoscopy the group over 70. In absolute numbers, the proportion was initiated. One showed no sign of enteritis, the other of C. difficile fatal cases (causative and contributive cases) was aborted because of bleeding and stenosis.
was 21 (<70) over 29 (>70), but in a relative way this was 4% over 6.5% or a 63% increase in the >70 age bracket. No Case reports
differences could be seen per 5 year age bracket >70. This clearly shows that the Salzburg cohort behaves as described From the 10 patients who received adjunctive probiotic in the literature (reviewed in Islam et al., 2012): relatively therapy 3 patients were selected to represent our case more CDI in older people and absolutely (and relatively) series population; these patients are discussed below. The more deaths contributed to or caused by C. difficile in >70 demographic characteristics, comorbidities, and clinical years. From this base line situation we selected the cases.
presentations of all patients are listed in Tables 3 and 4. None of this three cases experienced a relapse for more Patients characteristics, risk factors, and clinical
than six months, however, as described below, one of them (patient 1) had already been treated with a multistrain probiotic for a short time period before and had a relapse The demographic characteristics, comorbidities, and two weeks later.
clinical presentations of all patients are listed in Table 3. The mean age of the patients was 82 years (range 72- Patient 1 (Case 3 in Tables 3 and 4) 89); the majority were men (7 out of 10). All patients were hospitalised at the onset of CDI. Only one out of A 72 year-old male with tuberculosis, recurrent the 10 patients had no history of antimicrobials; 9 of 10 glomerulonephritis, and urinary tract infection (UTI) (due received antimicrobial medication (range 1 to 6, mean to extended spectrum β-lactamase producing Escherichia 3.7 different antibiotics) in the last three months before coli) who had been treated with hydrocortison and several onset of CDI. The most frequently administered antibiotics antibiotics (ciprofloxacin, rifampicin, amoxicillin+clavulanic were ciprofloxacin and amoxicillin+clavulanic acid (each acid and sulfonamide+trimethoprim) for six months was 5 out of 9 patients) and piperacillin+tazobactam (4 out of admitted to our hospital with the second relapse of CDI. 9 patients). 8 patients got PPIs and/or cortison before and He tested positive for C. difficile (ribotype 413) in the stool during the CDI-therapy. 4 out of 10 patients had received specimen taken at admission. After an initial treatment different single-strain probiotics before. All but one patient with metronidazole he was treated with vancomycin suffered from severe underlying diseases like malignancies, and a multistrain probiotic. Three days later, the patient renal failure or chronic vascular diseases. 6 of 10 patients experienced full clinical resolution. Even after further showed fever (defined as body temperature ≥38 °C at the treatment with ciprofloxacin and pivmecillinam for more time the stool samples were taken). Mean leukocyte count than a month, no CDI-relapse occurred.
was 13,500 (range 4,200 to 25,500) and mean CRP was 13.7 g/dl (range 3 to 26.2 g/dl).
Table 2. Population characteristics of Clostridium difficile infection cases at University Hospital Salzburg from 2009 to 2011.
% causative + contributive
Beneficial Microbes 4(1) Probiotics in Clostridium difficile infection Table 3. Demographic characteristics, underlying disease, manifestation of Clostridium difficile infection (CDI) (primary episode,
first or second relapse) and risk factors.
Primary CDI episode
Unknown antibiotic, Ciprofloxacin Ciprofloxacin, AmoxClav, Rifampicin, SulfTrim Cefazolin, PipTaz bladder carcinoma Ciprofloxacin, PipTaz, Ceftriaxon, SulfTrim, Vancomycin, AmoxClav bronchitis, recurrent urinary tract infection Ciprofloxacin, Levofloxacin, SulfTrim renal replacement therapy Ciprofloxacin, AmoxiClav, PipTaz endocarditis, CDI Clarithromycin, Moxifloxacin, Metronidazole peripheral arterial occlusive disease AmoxClav, Clarithromycin, Clindamycin, Ciprofloxacin, PipTaz sepsis, recurrent erysipelas, recurrent CDI AmoxClav, Meropenem, Metronidazole, Vancomycin, AmpSulbactam 1 PPI = proton pump inhibitors.
Table 4. Treatment, laboratory findings and outcomes of Clostridium difficile infection cases.1,2
Stool C. difficile
Repeated stool C. difficile
Fever ≥38 °C
Y, colon not described Y, colon not described Y, CT: known radiogenic Y, aborted, bleeding and 11.4 Y, colon not described Y, no sign of enteritis 12.11 16.86 complete 1 All patients received four times an oral dosage 125 mg vancomycin and twice daily a multistrain probiotic.
2 Abbreviations used: CRP = C-reactive protein; MSP = multistrain probiotic; MTZ = metronidazole; N = no; nd = not done; SSP = single strain probiotic; Y = yes.
3 Radiography with plain film, CT or sonography.
4 Patient died of pneumonia three months later.
Beneficial Microbes 4(1) M. Hell et al.
Patient 2 (Case 5 in Tables 3 and 4) An 85 year-old male was admitted to our hospital with Stool microbiota is best understood as a complex, living, a known bladder carcinoma and recurrent CDI with interdependent ecosystem. During periods of health, diarrhoea. He had already been tested positive for C. difficile bacterial gut residents suppress growth of C. difficile in in stool specimens one month before, after he had received the colon. Broad-spectrum antimicrobials have the potential ceftriaxon, and tested positive again in the stool specimen to disrupt the balanced ecology of the stool microbiota, taken at admission (ribotype 014). The first CDI episode creating an opportunity for C. difficile spores to germinate was treated with vancomycin and a multistrain probiotic. resulting in overgrowth and attendant production of toxins, The latter was prescribed for sixteen days, however, the which are responsible for most of the clinical symptoms patient was discharged from the hospital at day three. At of CDI and (pseudomembraneous)-colitis. Antibiotics like readmission, he was treated first with metronidazole for clindamycin are known to impair colonisation resistance, five days; after consulting an infectious disease specialist, however, second- and third-generation cephalosporins (for the therapy was switched to vancomycin, combined with which all clinical isolates of C. difficile are resistant) and a multistrain probiotic anew. Three days later, the patient fluoroquinolones are frequently used in hospital infections/ experienced full clinical resolution.
patients and cause iatrogenic CDI (Kelly, 2009). Patients can keep shedding (bacteria, toxins and spores) for some Patient 3 (Case 8 in Tables 3 and 4) weeks despite full clinical recovery. Those asymptomatic carriers emphasise the need for transmission reduction A 79 year-old male who underwent transcutaneous and universal infection control measures. Using gloves for aortic valve replacement developed endocarditis and was avoiding direct skin contact and hand hygiene with plain treated with clindamycin and ceftriaxon for one week, then soap and running water to remove spores followed by an followed by imipenem and moxifloxacin for 2 weeks. No alcoholic hand rub is preferred over alcohol-based hand diarrhoea was documented during this period nor stool rubs alone for symptomatic patients or patients with a samples were taken. One month later, after continously recent known CDI-episode. To reduce the environmental receiving moxifloxacin, he was readmitted with CDI, burden, spores should be removed with sporicidal agents reporting diarrhoea at home for already two weeks. (chlorine) on surfaces adjacent to the patients, as well as He tested positive for C. difficile (ribotype 053) in the for toilets and showers (Kelly 2009). Detergents based on stool specimen taken at admission and was treated with peroxides or glutaraldehyde are also effective (Tschudin- vancomycin and a multistrain probiotic. Five days later, the Sutter, 2012). The fundamental problem in CDI is not the patient experienced full clinical resolution.
presence of the pathogenic organism per se, but the absence of healthy microbiota to keep the growth of the pathogen Treatment and outcomes
suppressed. Accordingly, one would anticipate that the restoration of bacterial homeostasis in the colon could All patients under survey received 4× 125 mg oral resolve diarrhoea states caused by uncontrolled growth vancomycin and 2× the multistrain probiotic. Five patients of C. difficile.
suffered from recurrent CDI. Complete resolution of clinical presentation occurred in 9 patients (90%), and one of the C. difficile is a leading cause of AAD. The severity of observed subjects died within a 3-months follow-up period CDI ranges from mild cases, which require little more from pneumonia, apparently without a connection to the than the discontinuation of antimicrobials to intractable CDI episodes. No adverse events were reported. A repeated diarrhoea, to relapsing infection and severe life-threatening stool testing was performed in 9 out of 10 patients and illness. Mortality/fatality rates as high as 26% have been these proofed to be negative. Molecular characterisation of reported in old and very old patients, and patients with the strains was done in 70% (7 out of 10); PCR-ribotyping underlying disease like progressive solid tumours. Especially revealed thereby 5 different strains (2× 014, 2× 053 and haematology-oncology patients, having systemic diseases one of each 023, 433, 413) (Table 4). No clustering or and receiving high dose chemotherapy, are at risk for transmission was seen among the investigated patients.
CDI (Hautmann et al., 2011). Both the recurrence and overgrowth after initial dysbiosis can be partly explained Data from our surveillance system indicated 9 fatal courses by spore forming ability, specific adhesions in the colon directly related to CDI out of 151 cases (case-fatality ratio and hypervirulent strains via additional production of a 6%) during the observation period. 84 of the observed binary toxin. The cost per case ranges from approx. 2,500 patients were males; the mean age was 67 (range 19-94). USD (total cost estimate 3.5 billion) to 4,000 USD in the None of the fatal cases had a documented treatment with UK (Hickson, 2011), with health care system costs between 5,000 (USA) and 8,000 (EU) USD per primary episode and almost 14,000 USD for a case of recurrent CDI (Hautmann et al., 2011) with a total burden worldwide in the tens of Beneficial Microbes 4(1) Probiotics in Clostridium difficile infection billions. All available data show that CDI is much more Chapman, C.M., Gibson, G.R. and Rowland, I., 2011. Health benefits of difficult to prevent and cure than ordinary diarrhoea. In probiotics: are mixtures more effective than single strains? European addition, the spore forming ability results in enormous Journal of Nutrition 50:1-17.
Chinen, T. and Rudensky, A.Y., 2012. The effects of commensal microbiota on immune cell subsets and inflammatory responses. Recent meta-analysis on CDI treatment successes (Johnston Immunological Reviews 245: 45-55.
et al., 2012) in faecal transplantation in CDI therapy (Brandt, Cohen, S.H., Gerding, D.N., Johnson, S., Kelly, C.P., Loo, V.G., 2012; Van Nood et al., 2013) and this case series shows that McDonald, L.C., Pepin, J., Wilcox, M.H., the Society for Healthcare even in patients at high risk, with multiple severe underlying Epidemiology of America and the Infectious Diseases Society of diseases, administration of multistrain probiotics might be America, 2010. Clinical practice guidelines for Clostridium difficile beneficial by shortening the diseases course as well as by infection in adults: 2010 update by the Society for Healthcare preventing further relapses in patients with recurrent CDI. Epidemiology of America (SHEA) and the Infectious Diseases Furthermore, this paper presents a theoretical and practical Society of America (IDSA). Infection Control and Hospital basis to initiate well-designed clinical trials (Morrow et al., Epidemiology 31: 431-455.
2012) to evaluate multistrain probiotic treatment in CDI De Vuyst, L., Vrancken, G., Ravyts, F., Rimaux, T. and Weckx, S., 2009. patient groups with different underlying diseases.
Biodiversity, ecological determinants, and metabolic exploitation of sourdough microbiota. Food Microbiology 26: 666-675.
Dean, P., Maresca, M., Schuller, S., Phillips, A.D. and Kenny, B., 2006. Potent diarrheagenic mechanism mediated by the cooperative We would like to thank the heads of the different clinical action of three enteropathogenic Escherichia coli-injected effector departments at the University Hospital Salzburg, Paracelsus proteins. Proceedings of the National Academy of Sciences of the Medical University for their support with clinical data. The USA 103: 1876-1881.
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No financial conflicts of interest are declared for this study.
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Beneficial Microbes 4(1)
Fit for purpose? the facilitation directive and the criminalisation of humanitarian assistance to irregular migrants
DIRECTORATE GENERAL FOR INTERNAL POLICIES POLICY DEPARTMENT C: CITIZENS' RIGHTS AND CIVIL LIBERTIES, JUSTICE AND HOME AFFAIRS Fit for purpose? The Facilitation Directive and the criminalisation of humanitarian assistance to irregular migrants Abstract This study was commissioned by the European Parliament's Policy Department for Citizens' Rights and Constitutional Affairs at the request of the LIBE Committee. With renewed efforts to counter people smuggling in the context of an unprecedented influx of migrants and refugees into the EU, it assesses existing EU legislation in the area – the 2002 Facilitators' Package – and how it deals with those providing humanitarian assistance to irregular migrants. The study maps EU legislation against the international legal framework and explores the effects – both direct and indirect – of the law and policy practice in selected Member States. It finds significant inconsistencies, divergences and grey areas, such that humanitarian actors are often deterred from providing assistance. The study calls for a review of the legislative framework, greater legal certainty and improved data collection on the effects of the legislation.