Toxin production among epidemic Clostridioides difficile PCR ribotypes in Sweden

(1)

Örebro University

School of Medical Sciences

Independent project, 15 ECTS credits January 2020

Toxin production among epidemic

Clostridioides difficile PCR ribotypes in

Sweden

Author: Hanna Berghamre Supervisor: Torbjörn Norén Department of Laboratory Medicine Örebro University Hospital, Sweden

(2)

ABSTRACT Introduction

Clostridioides difficile infection (CDI) is the main cause of antibiotic microflora triggered enteritis. Production of toxin A and B elicit enterocytic inflammation and diarrhea. Hospital outbreaks from certain PCR ribotypes are well known due to excess morbidity and mortality. The toxin production of Clostridioides difficile is an important virulence factor, but it still remains unclear whether this capacity could explain the epidemic success of outbreak isolates.

Aim

The study aims to determine whether the amount of toxin production correlates to the pathogenicity of multiresistant Clostridoides difficile genotypes.

Materials and method

Nine strains of Clostridioides difficile were analyzed in regard to their respective toxin production. The epidemic strains have been obtained as part of the routine surveillance program by the National Reference Laboratory (NRL) in Örebro in 2012, 2013 and 2014. This retrospective study was done using a novel ELISA kit for quantitative detection of toxin A and B in solutions.

Results

The results show that the amount of toxin produced in vitro varies among the different strains. The highest concentrations of toxins were seen in ribotype 046 and 027, both of which are known to have caused outbreaks in Swedish hospitals.

Conclusion

It seems that the quantity of toxin production in Clostridioides difficile strains correlates well to their respective pathogenic potential. These findings could possibly encourage more research on the role of toxin production in C. difficile infections. The results could inspire investigation of future toxin-binding therapeutic candidates with little effect on the colonic microbiota.

(3)

Clostridioides difficile, formerly known as Clostridium difficile, is the main cause of antibiotic-associated diarrhea. C. difficile is a spore-forming, toxigenic bacteria. It is an obligate anaerobe, able to spread in aerobic conditions only in its spore form. Symptoms of C. difficile infections (CDI) range from mild diarrhea to pseudomembranous colitis. It can cause toxic megacolon and, in severe cases, infection might be fatal. Common additional symptoms are fever, abdominal pain and leukocytosis. Infections occur primarily in patients that are treated with antibiotics or have recently undergone such treatment. Antibiotics disrupt the normal colonic microflora and allows an overgrowth of resistant strains of C. difficile. In addition to antibiotic treatment, hospitalisation is an important risk factor for developing CDI, which is due to an increased risk of exposure to the pathogen. [1]

The incidence of CDI has decreased in Sweden over the last decade. Despite this, Sweden remains one of the countries in Europe with the highest incidence levels of CDI. [2] In 2018, an incidence of 63 cases per 100 000 inhabitants was reported. [3]

Infection with C. difficile can lead to increased mortality, longer time of hospitalisation and increased healthcare expenses. It has been shown that certain interventions can decrease the incidence of CDI in healthcare environments. These measures include restrictive prescription of antibiotics and improved procedures for infection control in hospitals. [4] Antibiotics such as cephalosporins and quinolones are known to significantly increase the risk of CDI. [5]

The first step in treating CDI is to end the current antibiotic regimen, if there is anyone ongoing. This intervention might give the colonic microflora a chance to recover and suppress the overgrowth of C. difficile. If this is not sufficient for recovery, patients are traditionally treated with metronidazole. Severe infections are treated with vancomycin. [6]

The ability to form spores is a crucial factor for the pathogenic success of C. difficile. Prior to manifest infection, patients can either be asymptomatic out-patient carriers, or become infected with the spores when hospitalised. After ingestion of the spores, certain bile acids in the intestine then act as germinants. These acids induce the transformation of the spores into metabolically active vegetative cells. [1]

(5)

2

Spore formation

Environmental cues indicating unfavorable conditions for the vegetative cells lead to activation of the transcription factor Spo0A, which is a key regulator of sporulation in C. difficile. Upon activation of Spo0A, a polar septum is created, forming a mothercell and a forespore. The forespore is then engulfed by the mothercell, after which the two cells cooperate in forming the mature spore. When this has been accomplished, the mothercell lyses and the spore is released. [1]

The spores of C. difficile are highly resistant to common cleansing preparations such as ethanol and hydrogen peroxide. The resistance to different decontamination methods varies among the strains. The only preparation that has been shown to consistently reduce C. difficile spores and vegetative cells on surfaces is hypochlorite (household bleach). [7]

Enterotoxins

Toxigenic strains of C. difficile produce two types of toxins, known as toxin A (TcdA) and toxin B (TcdB). In recent years, a third, binary toxin has been discovered, called CDT (Clostridioides difficile transferase). [8] At 308 kDa and 270 kDa respectively, Toxin A and B have the highest molecular weights of all known bacterial toxins.

TcdA causes an infiltration of neutrophils to the ileum. Along with TcdB, it brings on a disintegration of the intestinal epithelium by breaking up tight junctions between epithelial cells. Furthermore, the toxins influence GTPases which control the actin cytoskeleton of the epithelial cells. Altogether, these effects make the enterocytes undergo apoptosis.

TcdA and TcdB are glycosyltransferases, which influence the GTPases Rho, Rac and Cdc42. Inactivation of these enzymes by toxins such as TcdA and TcdB lead to severe disturbances in the normal function of the cell, due to a disruption of the actin cytoskeleton. Some strains, including the hypervirulent PCR ribotype 027, produce a third, binary toxin, called CDT (C. difficile transferase). CDT is an actin-specific ADP-ribosyltransferase which also has an important role in the pathogenesis of CDI. It causes an increased production of

proinflammatory cytokines, suppresses innate immune cells such as netrophils and disrupts the actin cytoskeleton. [9]

(6)

3

AIM

The study aims to determine whether the amount of toxin production correlates to the pathogenicity of multiresistant Clostridoides difficile genotypes

METHOD

Örebro University Hospital houses the National Reference Laboratory (NRL) for C. difficile. The laboratory has access to outbreak isolates and reference strains of C. difficile.

The selection of clinical strains

PCR ribotype (RT) 046 (human), 017 and 027 were collected from regional outbreaks in Sweden 2012 (Eksjö), 2014 (Ystad) and 2013 (Växjö) respectively. [2] RT 046 (pig) was sampled from a porcine outbreak in central Sweden. [10] RS338 (ribotype 017),RS42

(ribotype 001), RS392 (ribotype 012), RS394 (ribotype 038) and 078 were randomly selected from commercial in-house reference strains without epidemic reputation (Table 1).

PCR ribotype 046 caused an outbreak in Eksjö in 2012. [11]

The outbreak of PCR ribotype 017 in Ystad caused 27 established cases of CDI. Ten patients died due to the infection. [12] This PCR ribotype has a truncated toxin A gene and is

phenotypically toxin A negative. [13]

PCR ribotype 027 caused an outbreak in Växjö 2013. 24 patients were infected and four patients died due to the infection. [14]

(7)

4

Table 1. Summary of the C. difficile strains that were analysed.

1. 046 (human) Epidemic strain

2. 046 (pig) Isolate from porcine outbreak

3. 017 Epidemic strain

4. RS338 (ribotype 017) Reference strain

5. 027 Epidemic strain

6. RS42 (ribotype 001) Reference strain

7. RS392 (ribotype 012) Reference strain 8. RS394 (ribotype 038) Reference strain

9. 078 Reference strain

Laboratory procedure

The study was conducted using the tgcBIOMICS GmbH ELISA kit (Bingen, Germany) for quantitative detection of C. difficile toxin A and B in suspensions. A calibration curve was set up for each test, using the toxin positive control (standard control A or B, 80 ng/ml) diluted into eight different concentrations. Samples of the nine different strains (approximately 2 cm2 of each confluent agar plate) were suspended in 500 µl of dilution buffer. The diluted strains were then vortexed and centrifuged at 2500 G for four minutes.

100 µl of diluted standard control or 100 µl of supernatant from each of the centrifuged solutions were added in separate wells of the ELISA plate. 100 µl of dilution buffer was used as negative control. The plate was incubated in 36 ℃ for one hour. The wells were then washed three times using a dilution of the wash buffer and RO water (1+9). 100 µl of

conjugate (anti-toxin A or B) was added to each well and the plate was incubated in 36 ℃ for 30 minutes. The wells were washed three times. 100 µl of substrate was added to each well and the plate was left in room temperature for 15 minutes. 50 µl of stop reagent was added to each well. The plate was then analysed in a spectrophotometer, measuring the optical density (OD) at 450 nm and 620 nm.

(8)

5

Growth times of 24 and 48 hours were tested once for each toxin and strain. 48 and 72 hours were tested twice for each toxin and strain.

The toxin concentrations used for the standard curve were 40, 20, 10, 5, 2.5, 1.25, 0.65 and 0,31 ng/ml .

Ethical considerations

The bacterial strains that were tested had no traceable data from former individual patients. Only the genotypes of the strains were known. Consequently, the experiments on these strains do not pose any ethical issues regarding confidentiality or patient integrity.

RESULTS

For toxin B, there was a tendency for slower production compared to toxin A. The amounts of toxin B were very low after 24 hours and the concentration peaked for most strains at 72 hours (see Figure 2). Toxin A, on the contrary, reached high concentrations at only 24 hours of growth. Most strains reached a peak concentration at 48 hours and had a slight decline of toxin A concentration at 72 hours (see Figure 1).

The highest concentrations of toxin A were seen in 046 (1) and 027 (5) followed by RS42 (6). The same pattern could be observed for toxin B, where 027 had the highest production, closely followed by 046.

Epidemic PCR ribotype 017 (3) showed no production of toxin A. It did produce a relatively small amount of toxin B (see Figure 2).

(9)

6

Figure 1. Average of measured TcdA concentrations for each strain and growth time. Optical density is shown on the Y axis. Strains: 1. RT046 (human), 2. RT046 (pig), 3. RT017, 4. RS338, 5. RT027, 6. RS42, 7. RS392, 8. RS394, 9. RT078.

Figure 2. Average of measured TcdB concentrations for each strain and growth time. Optical density is shown on the Y axis. Strains: 1. RT046 (human), 2. RT046 (pig), 3. RT017, 4. RS338, 5. RT027, 6. RS42, 7. RS392, 8. RS394, 9. RT078. 0 0,5 1 1,5 2 2,5 3 3,5 1 2 3 4 5 6 7 8 9 Op tic al de ns ity Strains

Toxin A

24 hours 48 hours 72 hours

0 0,5 1 1,5 2 2,5 3 1 2 3 4 5 6 7 8 9 Op tic al de ns ity Strains

Toxin B

(10)

7

DISCUSSION AND CONCLUSION

The rise of multiresistant, hypervirulent strains such as PCR ribotype 027 is an urgent issue and a threat to patient security globally. The importance of toxins for the pathogenicity in hypervirulent strains of C. difficile is a debated subject among scientists. Some argue that toxins are not the most important virulence factor. [15] The results of this study points in the opposite direction, showing that the amount of toxin produced in vitro has a strong

correlation to the strains’ pathogenicity in vivo.

Epidemic PCR ribotype 017 did not show any significantly increased toxin production

compared to the reference strains. The strain did have a detectable production of toxin B after 72 hours of growth time, but only about half of the concentration of the other two epidemic strains (see Figure 2). The hypervirulence seen clinically in this strain might be due to a presence of the binary toxin CDT. Other explanations could be a slower toxin production, more potent toxins or highly efficient sporulation. To draw any conclusions, the strain should be tested in prolonged growth. PCR ribotype 017 has previously been shown to be toxin A-negative, which is in accordance with the results of this study. [13]

PCR ribotype 046 that was isolated from a porcine outbreak showed a much lower toxin production than PCR ribotype 046 that was sampled from a hospital outbreak in Eksjö. These results are noteworthy considering the fact that both strains are of the same ribotype. Perhaps the toxins of the porcine strains are more potent or pigs could be more sensitive to the effects of the toxins on enterocytes.

The non-epidemic strains generally showed low amounts of toxin production. An exception was RS42, which showed a significant production of toxin A after 48 hours of growth. RS42 is a commercial reference strain with no epidemic reputation.

To minimize the spreading of these potentially life-threatening pathogens, it is critical to implement effective measures for infection control and to restrict the prescription of antibiotics. Regarding infection control, the most important aspect is hand and surface hygiene. As C. difficile spores are not decontaminated by ethanol, it is necessary for healthcare professionals to thourougly wash their hands in addition to using hand sanitizer.

(11)

8

Concerning the sanitation of healthcare settings, surfaces contaminated with C. difficile should be cleaned with hypochlorite in order to eradicate the bacteria. [4]

In conclusion, this study shows that epidemic C. difficile strains 046 and 027 exhibit a high toxin production compared to the reference strains that were analyzed. This supports the view of toxins as a vastly important virulence factor for C. difficile, and also reinforces the notion that the amount of toxin production is a vital aspect for hypervirulence in this bacteria.

In addition to rational prescription of antibiotics and efficient infection control, more research is needed on the exact mechanisms of hypervirulence in C. difficile. All current available treatments have a disruptive effect on the intestinal microflora, which is an important risk factor for recurrence of CDI in patients. A thorough understanding of the pathogenesis is crucial for the development of new treatments, that preserve the colonic microbiota and in that way decrease the risk of relapse.

This was a small pilot study and it is therefore precarious to draw any definitive conclusions from the results. In order to do that, the analyses would need to be replicated and the strains could be tested in several other growth times than those present in this study.

ACKNOWLEDGEMENTS

I would like to thank my supervisor Torbjörn Norén who has inspired and guided me in the process of writing this paper. I would also like to thank my laboratory supervisor Eva Forsberg who has been of great help regarding the practical parts of the study.

(12)

9

REFERENCES

[1] Zhu D, Sorg JA, Sun X. Clostridioides difficile Biology: Sporulation, Germination, and Corresponding Therapies for C. difficile Infection. Front Cell Infect Microbiol; 8. Epub ahead of print 8 February 2018. DOI: 10.3389/fcimb.2018.00029.

[2] Rizzardi K, Norén T, Aspevall O, et al. National Surveillance for Clostridioides difficile Infection, Sweden, 2009-2016. Emerg Infect Dis 2018; 24: 1617–1625.

[3] Clostridioides difficile-infektion – sjukdomsstatistik — Folkhälsomyndigheten, http://www.folkhalsomyndigheten.se/folkhalsorapportering-statistik/statistik-a-o/sjukdomsstatistik/clostridium-difficile-infektion/ (accessed 20 January 2020). [4] Toepfer M, Magnusson C, Norén T, et al. [Insidious and widespread outbreak of

Clostridium difficile. Changed cleaning procedures and frequent evaluations cut infection rates in half]. Lakartidningen 2014; 111: 24–27.

[5] Price J, Cheek E, Lippett S, et al. Impact of an intervention to control Clostridium difficile infection on hospital- and community-onset disease; an interrupted time series analysis. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis 2010; 16: 1297–1302.

[6] Jarmo O, Veli-Jukka A, Eero M. Treatment of Clostridioides (Clostridium) difficile infection. Ann Med 2019; 1–9.

[7] Edwards AN, Karim ST, Pascual RA, et al. Chemical and Stress Resistances of Clostridium difficile Spores and Vegetative Cells. Front Microbiol 2016; 7: 1698. [8] Gerding DN, Johnson S, Rupnik M, et al. Clostridium difficile binary toxin CDT. Gut

Microbes 2014; 5: 15–27.

[9] Chandrasekaran R, Lacy DB. The role of toxins in Clostridium difficile infection. FEMS Microbiol Rev 2017; 41: 723–750.

[10] Norén T, Johansson K, Unemo M. Clostridium difficile PCR ribotype 046 is common among neonatal pigs and humans in Sweden. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis 2014; 20: O2-6.

[11] Läkartidningen - Lömskt och omfattande utbrott av Clostridium difficile,

http://www.lakartidningen.se/Klinik-och-vetenskap/Vardutveckling/2014/01/Lomskt-och-omfattande-utbrott-av-Clostridium-difficile/ (accessed 15 January 2020).

[12] Loftrup-Ericson I. Usel hygien bakom bakterieutbrott. SVT Nyheter, 29 October 2014, https://www.svt.se/nyheter/lokalt/skane/usel-hygien-bakom-bakterieutbrott (29 October 2014, accessed 17 January 2020).

[13] Cairns MD, Preston MD, Lawley TD, et al. Genomic Epidemiology of a Protracted Hospital Outbreak Caused by a Toxin A-Negative Clostridium difficile Sublineage PCR Ribotype 017 Strain in London, England. J Clin Microbiol 2015; 53: 3141–3147.

(13)

10

[14] Utbrott av Clostridioides difficile i Växjö — Folkhälsomyndigheten,

https://www.folkhalsomyndigheten.se/nyheter-och-press/nyhetsarkiv/2014/januari/utbrott-av-clostridium-difficile-i-vaxjo/ (accessed 15 January 2020).

[15] Merrigan M, Venugopal A, Mallozzi M, et al. Human hypervirulent Clostridium difficile strains exhibit increased sporulation as well as robust toxin production. J Bacteriol 2010; 192: 4904–4911.

[16] Lee H-Y, Hsiao H-L, Chia C-Y, et al. Risk factors and outcomes of Clostridium difficile infection in hospitalized patients. Biomed J 2019; 42: 99–106.

[17] Yakob L, Riley TV, Paterson DL, et al. Mechanisms of hypervirulent Clostridium difficile ribotype 027 displacement of endemic strains: an epidemiological model. Sci Rep 2015; 5: 1–9.

[18] Utbrott av Clostridioides difficile i Växjö — Folkhälsomyndigheten,

http://www.folkhalsomyndigheten.se/nyheter-och-press/nyhetsarkiv/2014/januari/utbrott-av-clostridium-difficile-i-vaxjo/ (accessed 21 December 2019).

[19] Berry CE, Davies KA, Owens DW, et al. Is there a relationship between the presence of the binary toxin genes in Clostridium difficile strains and the severity of C. difficile infection (CDI)? Eur J Clin Microbiol Infect Dis Off Publ Eur Soc Clin Microbiol 2017; 36: 2405–2415.

[20] Sukumar MR, König B. Pomegranate extract specifically inhibits Clostridium difficile growth and toxin production without disturbing the beneficial bacteria in vitro. Infect Drug Resist 2018; 11: 2357–2362.

[21] Papatheodorou P, Barth H, Minton N, et al. Cellular Uptake and Mode-of-Action of Clostridium difficile Toxins. Adv Exp Med Biol 2018; 1050: 77–96.

[22] Vardakas KZ, Konstantelias AA, Loizidis G, et al. Risk factors for development of Clostridium difficile infection due to BI/NAP1/027 strain: a meta-analysis. Int J Infect Dis IJID Off Publ Int Soc Infect Dis 2012; 16: e768-773.

[23] Läkartidningen - Lömskt och omfattande utbrott av Clostridium difficile,

http://www.lakartidningen.se/Klinik-och-vetenskap/Vardutveckling/2014/01/Lomskt-och-omfattande-utbrott-av-Clostridium-difficile/ (accessed 15 January 2020).

(14)

11 24 hours 48 hours 72 hours 1 2,995 3,10967 3,088 2 0,296 1,19133 0,764 3 0,095 0,05716 0,078 4 0,146 0,08067 0,0935 5 3,14 3,129 2,996 6 0,667 3,01067 2,8815 7 0,127 1,928 1,815 8 0,263 0,126 0,0745 9 0,146 0,094 0,099

Figure 1. Average values per strain for Toxin A

24 hours 48 hours 72 hours 1 0,085 1,26067 2,148 2 0,061 0,093 0,126 3 0,144 0,855 0,8215 4 0,145 0,65967 1,1585 5 1,284 2,39933 2,4035 6 0,121 0,463 0,518 7 0,09 0,187 0,241 8 0,09 0,10933 0,1505 9 0,113 0,10333 0,309