# Presentation: 146266

## German Experience with non-O157:H7 STEC

**German Experience with ****non-O157:H7 STEC**

**Martina Bielaszewska**
**Institute of Hygiene, and**
**The National Consulting Laboratory on Hemolytic Uremic Syndrome**
**University of Münster**
**Münster**
** ****Germany**** **

**The USDA/FSIS meeting „The Public Health Significance of non-O157 Shiga Toxin-Producing *****E. coli***** (STEC)“**
**Washington, D.C., October 17, 2007**

## Notifiable microorganisms according to § 7of the German Protection against Infection Act 

**Notifiable microorganisms according to § 7****of the German Protection against Infection Act**

**1.****	****Adenovir****uses**
**2.****	****Bacillus anthracis**
**3.****	****Borrelia recurrentis**
**4.****	****Brucella sp.**
**5.****	****Campylobacter sp.**
**6.****	****Chlamydia psittaci**
**7.****	****Clostridium botulinum ****or toxin detection**
**8.****	****Corynebacterium diphtheriae, ****t****oxi****genic**
**9.****	****Coxiella burnetii**
**10.****	****Cryptosporidium parvum**
**11.****	****Ebolavirus**
**12.****	****a) STEC/EHEC**
**	****b) E. coli, ****other enteropathogenic**
**13.****	****Francisella tularensis**
**14.****	****FSME-Virus**
**15.****	****Yellow fever ****virus**
**16.****	****Giardia ****L****amblia**
**17.****	****Haemophilus influenzae**** (liquor, blood)**
**18.****	****Hantavir****uses**
**19.****	****Hepatitis**** ****A**** v****irus**
**20.****	****Hepatitis**** ****B**** v****irus**
**21.****	****Hepatitis**** ****C**** v****irus**** (not chronic i****nfe****c****tion****)**** **
**22.****	****Hepatitis**** ****D**** vi****rus**
**23.****	****Hepatitis**** ****E**** v****irus**
**24.****	****Influenza**** v****ir****uses (only direct evidence)**

**25.****	****Lassavirus**
**26.****	****Legionella sp.**
**27.****	****Leptospira interrogans**
**Listeria monocytogenes**** (blood, liquor, newborns)**** **
**Marburgvirus**
**30.****	****Measles ****virus**
**31.****	****Mycobacterium leprae**
**32.****	****Mycobacterium tuberculosis/africanum, Mycobacterium bovis**
**33.****	****Neisseria meningitidis**** (liquor, blood)**** **
**34.****	****Norwalk-****like v****irus**** (stool)**
**35.****	****Poliovirus**
**36.****	****Rabiesvirus**
**37.****	****Rickettsia prowazekii**
**38.****	****Rotavirus**
**Salmonella Paratyphi**** (direct evidence)**** **
**Salmonella Typhi**** (direct evidence)**
**41.****	****Salmonella, ****others**
**42.****	****Shigella sp.**
**43.****	****Trichinella spiralis**
**44.****	****Vibrio cholerae O 1 und O 139**
**45.****	****Yersinia enterocolitica**
**46.****	****Yersinia pestis**
**47.****	****Other causes of haemorrhagic fever**

## Suspicion, disease and death § 6 Abs.1 Nr.1  

**Notifiable Diseases ****according to § 6****of the German Protection against Infection Act**** **

**Enteropathic haemolytic-uraemic syndrome (HUS)**
- Botulisms
- Cholera
- Diphtheria
- Humane spongiform enzephalopathy
- Acute viral hepatitis
- g)  Viral haemorrhagic fevers

- h)	Measles
- i)	Meningococcal meningitis or -septicaemia
- j)	Anthrax
- k)	Poliomyelitis
- l)	Plague
- m) Rabies
- n)	Typhus abdominalis/
-     Paratyphus
- Disease and death from tuberculosis

- Suspicion, disease and death § 6 Abs.1 Nr.1

**Notes:**
Dies sind die in § 6 Nr. 1 aufgeführten Krankheitsbilder, die einheitlich bei Verdacht, Erkrankung und Tod meldepflichtig sind. Es entfallen Untergruppen, in denen z.B. nur der Tod, wie vorher bei Masern, oder nur Erkrankung und Tod, aber nicht Verdacht, wie bei einer Reihe von Krankheitsbildern meldepflichtig ist. 

## Dual communication flow according to the German Protection against Infection Act 

- Physician

- Public Health Office

- Robert Koch Institute

- Laboratory

**Dual communication flow according to the German Protection against Infection Act **

**Analysis of epidemiological data**
**Feedback of data**
**Interventions**

**Control of data validity**
**Local action**

## Reported STEC infections and HUS cases

**Reported STEC infections and HUS cases**
**  ****in Germany (2001-2006)  **

- Epidemiol. Bulletin Robert Koch Institute

**Year**

**Number**

-  80% of reported STEC are non-O157

## Risk factors for STEC infections in Germany

**Risk factors for STEC infections in Germany**

- Laboratory-based sentinel surveillance study in 14 of 16 German federal states  (April 2001-March 2003)
-  202 cases (3 mo-89 yrs, median 2.5 yrs) (_86% non-O157 infection_, 5 HUS)
-  202 age- and region-matched controls
**Age-specific risk factors**
- <3  yrs:  direct contact with ruminants
- 	 playing in sandbox
-               drinking raw milk
- 3-9 yrs:	  playing in sandbox
- > 10 yrs: eating lamb meat
-   eating raw spreadable sausages

- Werber et al., Am. J. Epidemiol. 2007

## STEC serogroups

**STEC serogroups**

- Werber et al., Am. J. Epidemiol. 2007

- * Abdominal pain, nausea, vomiting,
-   fever, blood in stool, or incomplete clinic

## Prevalence of STEC serotypes in sporadic cases of HUS in Germany (1996-2006) 

**Prevalence of STEC serotypes in sporadic cases ****of HUS in Germany (1996-2006)**** **

**(n = 787)**

**17.3%**

**13.2%**

**7%**

**2.5%**

**6.4%**

** ****3.4%**

- 50.2%

- Bielaszewska et al., PLoS ONE, 2007

- Others
- 6.4%

-     **O111:H8/H-**
**2.5%**

**O103:H2**

## Outbreaks caused by SF STEC O157:H-in Germany

**Outbreaks caused by SF STEC O157:H****-****in Germany**

**1995/96 (12-03)****	****     Bavaria****	****          28****	****    3****	****      Teewurst***
**2002 (03)****	****     Münsterland           6****	****    0****	****      Unknown**
** ****(10-12) ****	****     Bavaria, B-W         38 ****	****    4****	****      Apple juice***
**	****	****	****	****	****	****	****	****Curdled cheese* **
**2006 (04-08)****	****    North Germany      18                0            Unknown**

-   **Year (mo)****	****       Location****	****        HUS          Deaths****	**** ****Case-control study**
**	****	****	****	****      ****cases (n)****	****    (n)        **

- Ammon et al., J. Infect. Dis. 1999; Epidemiol. Bull. of Robert Koch Institute, 2002, 2003; Fruth et al., 3rd International
-  Workshop on Thrombotic Microangiopathies, Jena, Germany, October 2007

- * SF STEC O157 never isolated from any food or environmental samples

## Sorbitol fermentation	 	+	    -	          	 

**Sorbitol fermentation****	**** ****	****+****	****    -****	****          ****	**** **
******-Glucuronidase ****	**** ****	****+****	****    -****	****          **
**Motility****	**** ****	****-****	****    +****	**
**Seasonality****	****   Winter ****	****Summer**
**Age of patients****	****  < 3 yrs****	****> 3 yrs**
**Reservoirs****	****  Unknown****	****Ruminants ****	**
**Transmission routes****	****  Unknown****	****Food, water, ****	****	****	****	****direct contact **
**Risk for HUS****	****        ****?****	****   ******** 10****%**
**	****	****	**** ****	**
**	****	**
- 		,

**Differences between SF STEC O157:H****-**
**and STEC O157:H7**

- Karch & Bielaszewska, J. Clin. Microbiol., 2001; Karch et al., Int. J. Med. Microbiol., 2005; Tarr et al., Lancet, 2005; Werber et al., Am. J. Epidemiol. 2007

**STEC O157:H7**

**SF STEC O157:H****-**

## SF STEC O157:H- are spreading!

**SF STEC O157:H****-**** are spreading!**

**Countries with reported isolation of SF EHEC O157:H****-**** (2007)**

**Austria**

**Australia**

**South Korea**

**Finland**

## Outbreaks caused by non-O157 STEC

**Outbreaks caused by non-O157 STEC**

**Serotype   ****	****Year        Settlement    ****	****     Cases/HUS        Source**
**O22:H8****	****   ****	****1989****	****    family****	****             3/1****	**** cattle (milk)**
**O26:H11****	****1999****	****    community****	****             3/3             unknown**
**O26:H11     ****	**** 2000****	****    day care centres,        11/0           Seemerrolle* ****	****	****                                 hospitals (3 states)****	****	****       (beef)               **
**O111:H8****	****1986****	****     family****	****              4/1****	****  unknown**
**O145:H28****	****1999****	****     family                           2/0****	****  unknown**
**	****	****    ****	****2001****	****     kindergarten                6/1****	****  unknown**
**	****	****    ****	****2002           family                            2/0            unknown**
*** Identified as a possible source based on comparison of invoices of purchased food, but the strain not isolated from the food**
**       **
-         Bockemühl et al. Bundesgesundhbl. 1990; Misselwitz et al., Pediatr. Infect. Dis., 2003; Sonntag et al., J. Clin. Microbiol., 2004; Werber et al., J. Infect. Dis. 2002

## STEC O26

**STEC O26**

**The most frequent non-O157 STEC isolated from HUS patients ( > 1/3 of non-O157 STEC, 13.2% of all STEC)**
**Serotypes O26:H11 and O26:H****-****, both possess *****fliC*****H11**
**Stx production: Stx1, Stx2, Stx1+Stx2**
**Temporal shift from *****stx*****1**** to *****stx*****2 ****since 1990th**
**The *****stx*****2**** genotype is associated with HUS **
**    ****(*****P *****< 0.001) **

- Zhang et al., J. Clin. Microbiol. 2000
- Bielaszewska et al., J. Clin. Microbiol. 2005

## STEC O145

- Sonntag et al., J. Clin. Microbiol. 2004

-  Two different serotypes (*fliC* types)
-  Two different* eae* types
-    O145:H28 (98%), *eae* 
-    O145:H25 (2%), *eae* 
-   5 *stx* genotypes
*   *
*    **stx*2 		(70.8%)
*    **stx*1 		(21.7%)
*    **stx*1+ *stx*2 	(2.5%)
-     *stx*2c 		(4.2%)
*    **stx*1+ *stx*2c 	(0.8%)

**STEC O145**

** ****Second most  frequent HUS-associated non-O157 STEC (7%)**

## STEC O111

**STEC O111**

- Zhang et al., Int. J. Med. Microbiol., 2007

## Subtyping scheme for STEC O111 

**Subtyping scheme for STEC O111**** **

**Serotype****	*****fliC******	******stx******	******eae*****	****      OI-122****	*****cad***** genes       LDC***
**  ****	****	****	****	****	****            ****(*****efa-1/pagC/sen*****)      (A/B/C)**
**O111:H8/H****-****	****H8       1,2,1+2****	****   +****	****      +/+/+****	**** ****	****      -/+/-****	****	****-**
**   ****(n = 64)**
**O111:H10/H****-****	****H10****	**** 2****	****   -****	****        -/-/-****	****  ****	****      +/+/+****	****              +**
**    ****(n = 5)**
**O111:H11/H****-****	****H11****	**** 1****	****   +****	****       +/+/+****	****      +/+/+****	****	****+**
**    ****(n = 3)**

- Zhang et al., Int. J. Med. Microbiol., 2007

*** Lysin decarboxylation**

## stx genes in STEC O111:H8/H-

***stx***** genes in STEC O111:H8/H****-**

- Zhang et al., Int. J. Med. Microbiol., 2007

- 0

- 5

- 10

- 15

- 20

- 25

- 1955-1989

- 1990-1999

- 2000-2005

*stx*1

*stx*2

*stx*1*+stx*2

** ****Stx production: Stx1, Stx2, Stx1+Stx2**
** ****Temporal shift from *****stx*****1**** to *****stx*****1*****+stx*****2**** since 2000 **

** ****The introduction of *****stx*****2**** is associated with HUS **
**    ****(*****P *****< 0.001)**

**Year of isolation**

**No. of isolates**

## Production of Stx2dactivatable is a predictor for a severe 

**Production of Stx2d****activatable**** is a predictor for a severe **
**outcome of infections caused by *****eae*****- STEC**

| Clinical
outcome | Total isolates  | No. (%) of 
eae- | No. (%) of eae-negative STEC producing |  | Clinical associations of Stx2dactivatable-producing STEC with outcome |  |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  | Stx2dactivatable* | non-activatable Stxs ** | Outcome | P |
| HUS | 337 | 11 (3) | 7 (64) | 4 (36) | HUS vs. others | < 0.00001 |
| BD | 50 | 6 (12) | 6 (100) | 0 (0) | Severe (HUS+ BD) vs. milder (D) disease | 0.0001 |
| D | 397 | 113 (29) | 17 (15) | 96 (85) | BD vs. D | 0.0009 |
| A | 108 | 79 (73) | 0 (0) | 79 (100) | Disease vs. A | < 0.0001 |

- Bielaszewska et al., Clin. Infect. Dis. 2006

*** Most common serotypes O91:H21, O113:H21**
**** ****Stx1, Stx1c, Stx1d, Stx2, Stx2c, Stx2d****EH250****, Stx2e, Stx2f**

** ****Rapid and comprehensive subtyping of *****stx***** genes in STEC isolates **
**   ****is necessary to alert a clinician about the risk of HUS development**

## Diagnostic scheme for the detection 

- STOOL SAMPLE

- Screening

- Results

- Interpretation

- Action

***eae***

*** ******sfpA***

***rfb******O157***

***stx***

**Culture**
- - SMAC
- - CT-SMAC
- - EHEC-Hly agar
** ****Isolation**
- - Char. phenotypes
- - Colony blot
-    hybridization
**Characterization**
-  _Molecular_
-   - *stx* subtyping
-   - virulence genes
- _Phenotypic_
- - Stx production
- - EHEC-Hly
- - Te-resistance

- Targets

**Diagnostic scheme for the detection **
**of STEC in stools**

- Adapted from Friedrich et al., J. Clin. Microbiol. 2004

**Non-O157 ****STEC (*****eae*****+)**

***stx+/eae+/rfb*****O157*****+/sfp*****A*****-***

***stx+/eae+/rfb*****O157*****+/sfp*****A*****+***

**    ****STEC O157:H7**

**   ****SF STEC O157:H****-**

**Non-O157 ****STEC **
**(*****eae*****-)**

## Nucleotide sequence analysis of pSFO157

- Brunder et al. Int. J. Med. Microbiol. 2006

- Schmidt et al., Infect. Immun. 1995
- Brunder et al., Microbiology 1996
- Brunder et al., Mol. Microbiol. 1997
- Burland et al., Nucleic Acids Res. 1998

**Nucleotide sequence analysis of pSFO157**

- Brunder et al., Infect. Immun. 2001
- Brunder et al., Int. J. Med. Microbiol. 2006

## Diagnostic scheme for the detection 

- STOOL SAMPLE

- Screening

- Results

- Interpretation

- Action

***eae***

*** ******sfpA***

***rfb******O157***

***stx***

**Culture**
- - SMAC
- - CT-SMAC
- - EHEC-Hly agar
** ****Isolation**
- - Char. phenotypes
- - Colony blot
-    hybridization
**Characterization**
-  _Molecular_
-   - *stx* subtyping
-   - virulence genes
- _Phenotypic_
- - Stx production
- - EHEC-Hly
- - Te-resistance

- Targets

**Diagnostic scheme for the detection **
**of STEC in stools**

- Adapted from Friedrich et al., J. Clin. Microbiol. 2004

**Non-O157 ****STEC (*****eae*****+)**

***stx+/eae+/rfb*****O157*****+/sfp*****A*****-***

***stx+/eae+/rfb*****O157*****+/sfp*****A*****+***

**    ****STEC O157:H7**

**   ****SF STEC O157:H****-**

**Non-O157 ****STEC **
**(*****eae*****-)**

## Isolation of STEC O157- SMAC

**Isolation of STEC O157- SMAC**

**     **

**Non-O157 STEC**

## Isolation of non-O157 STEC

**Isolation of non-O157 STEC**

**EHEC hemolysin is expressed ****	**
**   ****O26, O103, O111, O145 = *****eae*****+ **
**	****O91:H21, O113:H21 = *****eae*****-**
**                      **

- Bielaszewska et al., J. Clin. Microbiol. 2005
- Mellmann et al., Clin. Infect. Dis. 2005
- Sonntag et al., J. Clin. Microbiol. 2004
- Zhang et al., Int. J. Med. Microbiol. 2007
- Bielaszewska et al., PLoS ONE 2007

**Isolation on enterohemolysin agar**

** ****EHEC hemolysin not expressed**
**    ****SF EHEC O157:NM**

**Isolation on SMAC (not CT-SMAC   tellurite-sensitive!)**
- -** Slide agglutination**
** ****Colony hybridization ****(*****stx*****, *****eae***** probes)**

**Selective/diagnostic medium needed**

## Isolation of STEC strains is a requisite, not a luxury!

**Isolation of STEC strains is a requisite, not a luxury!**

**Centers for Disease Control and Prevention (CDC).**
**Importance of culture confirmation of Shiga toxin-producing *****Escherichia coli***** infection as illustrated by outbreaks of gastroenteritis-New York and North Carolina, 2005.**
- MMWR Morb Mortal Wkly Rep. 2006;55(38):1042-5.

_**Isolation of STEC **_**from stools is **_**necessary**_** for:**
** ****Epidemiological studies **
** ****Public health actions**
** ****Monitoring of virulence**
** ****Emergence of highly pathogenic clones**

## Acknowledgment

**IFH Münster**
**Helge Karch**
**Alexander Friedrich**
**Alexander Mellmann**

**Innsbruck**
**Bernd Zimmerhackl**

**Stockholm**
**Andrea Ammon**

**RKI Berlin**
**Dirk Werber**
**Katharina Alpers**

**RKI Wernigerode**
**Angelika Fruth**
**Rita Prager **
**Helmut Tschäpe**

**Acknowledgment**