clostridium difficile: a new

“Clostridium difficile: a new foodborne pathogen?”

Patrizia Spigaglia

Ist ituto Superiore di Sanità - Rome, Italyp a t r i z i a . s p i g a g l i a @ i s s . i t

TO.BA.FOOD - TOxingenic BActeria in FOOD - 9th November 2017 – Turin, Italy

Clostridium difficile

Symptomless carriership

5% healthy adults

> 30% hospidalized patients

Infection (CDI)

Range from mild diarrhea to life threatening disease(pseudomembranous colitis, toxic megacolon)

C. difficile is a gram-positive, spore-forming, anaerobic bacterium recognized as the major cause of healthcare antibiotic-associated diarrhea


Risk factors

• Antibiotic exposure

• Age >65 years

• Hospitalization or long-term care facility exposure

• Comorbidities (malignancy, cystic fibrosis, inflammatory bowel disease, diabetes mellitus, cirrhosis, chronic kidney disease, immunodeficiency, solid organ or hematopoietic stem celltransplantation)

• Chemotherapy, immunosuppressants, proton pump inhibitors

• Gastrointestinal surgery


CMI.2001. 7: 411- 416

Pathogenesis of CDI

• Infection occurs after disruption or alteration of the intestinal microbiota

• There are over 400 different types of C. difficile strains

• Only strains producing Toxin A and Toxin B cause disease


• 453 000 CDI / year

• 29 500 deaths / year

• 1st cause of HAI in U.S. (12.5%)

• 123 997 CDI / year

• 9% mortality

• 8th cause of HAI in EU (5.4 %) http://ecdc.europa.eu/en/publications/Publications/healthcare-associated-infections-antimicrobial-use-PPS.pdf

USA

Europe

Burden of CDIIncrease in incidence and severity of CDI has been related to the emergence of highly virulent

(HV) strains (RT 027 and RT 078)

N Engl J Med 2015;372:825-34


• Produce higher levels of Toxin A and B

• Produce the binary toxin (CDT)

• Sporulate earlier and with greater efficiency

• Resistance to multiple classes of antibiotics (MDR)

HV strains (RT027 and RT078) vs Historic


Toxin A and B acts by modifying host cell GTPase proteins

CDT induces formation of long microtubule-based protrusions at the surface of intestinal epithelial cells

Microbiol. Mol. Biol. Rev. 2013. 77 :567-581

Toxicon. 2012. 60, 572-581; JMM. 2013.62:1486-1489;

Emerg Infect Dis. 2011. 17:976-982; Gut Microbes. 2014. 5: 15–27.

Journal of Bacteriology. 2010. 192 : 4904-4911

Ther Adv Infect Dis. 2016. 3: 23-42

Community acquired CDI (CA-CDI)

Increased incidence of CDI in the community

34 EU countries: 14%

US multi-centre study: 32%

Many CDI cases without an apparent link to health care

The HV RT 078 is the prevalent cause of CA-CDI

Lancet 2011; 377: 63–73

Infect Dis Clin N Am 29 (2015) 29–35


C. difficile in animalsC. difficile has been detected in several animal species and also recognized as cause of infection

• Food-producing animals (e.g. pigs and cattle)

• Companion animals (e.g. horses and dogs)

• Laboratory animals (e.g. hamsters and guinea pigs)

• Wildlife in captivity (e.g. ostriches and elephants)

In North America CDI is considered the most significant cause of neonatal diarrhoea in swine

Outbreaks of CDI have been reported in companion animals


Most of the RTs detected in animals are cause of infection in humans

RTs in animals

BMC Microbiol 2012. 12: 48; BMC Microbiol 2014. 14: 173; Adv Exp Med Biol 2016. 932:65-92

Prevalence of C. difficile RT 078 in food animals

RT 078 is the prevalent type detected in food-producing animals

RT 078 is predominant in animals


WGS and phylogenetic analysis on 65 isolates RT078

Farmers and pigs were colonized with identical C. difficile RT 078 clones strongly suggesting that an interspecies

transmission has occurred

Zoonotic transmission of HV RT 078 seems possible


C. difficile has been detected in various foods including:

• Meat (ground beef / veal / pork, chicken meat )

• Fishes, seafood, molluscs

• Vegetables

C. difficile in foods


Meat

Journal of Applied Microbiology. 2016. 122: 542-553

RT 078 is the prevalent type


Infect Dis Clin N Am 2013. 27: 675-685

USA and Canada: 6 - 44%

Europe: < 3%

C. difficile prevalence rate

Clin Microbiol Infect 2016; 22: 266.e1–266.e7

Europe and USA 0.5 - 20.7% calves slaughtered between 21- 27 weeks

Australia 10 - 58 % calves slaughtered between 7–14 days

20% at 0–16 days

0.7% at 90–120 days

0% at 150 days

The age of slaughter is a significant and perhaps underappreciated risk factor for carcasses

contamination during the slaughter process


MeatC. difficile colonization in calves :

C. difficile prevalence in carcasses of calves :

Seafood and fish5% of the 119 seafood and fish investigated samples (including ready-to-

eat shrimp) were positive for C. difficile RT 078

The source of contamination :

- carriage of C. difficile

- water contamination

- cross-contamination during processing and sale


Molluscs• 4 % of the 925 investigated samples were C. difficile-positive

• The most frequent RT was 078/126

• 19.4% resistant to MXF

30.5% resistant to CLI

38.8% resistant to ERY

100% resistant to CIP


Vegetables• 2.4 – 8.0 % positive for C. difficile

• RTs: 078, 027, 001, 014/020, 015

CD survives sewage treatment and can be recovered in high prevalence in biosolids

CD persists over a 9-month period when biosolids is applied to land

CD grows within the subsurface of biosoild-amended soil


• Resistance to MXF and CLI is frequent in strains isolated from vegatables

Journal of Applied Microbiology. 2016. 122: 542-553

Biosolids

Water

Growth of C. difficile

Conditions for optimal C. difficile growth:

• pH range of 7 – 9

• 30°C - 37C° (growth T range: 25C°- 45°C)

• Strictly anaerobic conditions (H2 10% - CO2 5% - N2 85%)

Journal of Applied Microbiology. 2016. 122: 542-553 Foodborne Pathog Dis 12, 177–182


Growth of C. difficile

Journal of Applied Microbiology. 2016. 122: 542-553 Foodborne Pathog Dis . 2015 12: 177–182

Growth limits (RT 078 and 027):

Reduced growth at pH < 6.5 and salt concentration > 4%

No growth between 4 and 21 C°

Inhibited growth by preservatives commonly applied to control clostridia (nitrite, nitrate, sodiummetabisulphite and nisin)


• The control measures adopted for the other clostridia would also be effective against C. difficile

• Spores rather than vegetative cells need to be ingested to cause CDI

Spores


C. difficile spores are resistant to chemical antimicrobials

5 log CFU reduction requiring chlorine concentrations of over 3000 ppm for

15 min or 30% v/v hydrogen peroxide vapor

C. difficile spores are resistant to heat

High level of inter-strain variation: D value (time for 10-fold reduction in

viable numbers) at 100 C° varies from 2.5 to 33 min

Spores

C. difficile spores can survive recommended cooking practices (internal T of 70°C for up 2 min)

Spores from RT 078 seem to have enhanced heat tolerance compared to those of other RTs


Discrepancies in C. difficile prevalence

Detection of C. difficile in food


There are currently no published data addressing

the best methods for detection of C. difficile in foods

Sodium taurocholate bile salt

Lysozyme

Enrichment stepIngestionof spores Bile or bile breakdown products are

necessary for spores germination

When the microflora is disrupted the bile acids (cholate, taurocholate and glycocholate) are not metabolized thereby activating germination

Removal of the exosporium results in an increase of colony formation

The addition of blood to culture media may be a source of lysozyme

Journal of Applied Microbiology. 2016. 122: 542-553 Foodborne Pathog Dis. 2015. 12: 177–182


Common steps in C. difficile detection

Centrifugation, pellet streaking onto on agar plates (CDMN, CCFA) Incubation for 48h at 37°C in anaerobic conditions Toxins detection by EIA or NAAT methods


Enrichment Culture in pre-reduced broth (usually C. difficile-moxalactam-norfloxacin -

CDMN) + 5% horse blood + 0.1% sodium taurocholate Incubation at 37°C for 7-10 days in anaerobic conditions

Alcohol (or Heat) shock

Culture broth + 96° ethanol (1:1 [vol/vol]) at room temperature for 50 min(or 80°C 10 min)

Isolationand

toxins detection

Typing Capillary gel electrophoresis based PCR-ribotyping

Infectious dose

Direct plating method of retail ground beef and pork indicate that the number of spores is low:

30 spores/g (range 20–60 spores/g) for pork

100 spores/g (range 20–240 spores/g) for beef

Enrichment step makes difficult to quantitatively assess the burden of C. difficile spores

The infectious dose of C. difficile spores / vegetative cells is currently unknown


At-risk population

• Inadequate immune response

• Treatment with proton inhibitors

(reduced acidity remodel the microbiome thereby increasing susceptibility to CDI )

• Treatment with antibiotics

(the bile acids are not metabolized thereby supporting C. difficile outgrowth)

Vulnerable population for potential foodborne transmission of C. difficile have to be defined

Clin Infect Dis. 2010. 51, 577–582Journal of Applied Microbiology. 2016. 122: 542-553 Foodborne Pathog Dis 12, 177–182


• No reported cases of food-associated CDI

• Standardization of sampling, culturing and identification methods

• Determine the infective dose of C. difficile for humans

• Define potential at-risk population

Points to be clarified


Food is a potential source of C. difficile in the community

“CDI sits firmly under the One Health umbrella, in which human health, animal health and the environment are inextricably linked”


clostridium difficile: a new

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