Molecular epidemiology of community- and hospital-associated Clostridioides difficile infections in Jönköping, Sweden, October 2017 - March 2018.

Clostridioides difficile infections (CDIs) in Sweden are mostly hospital-associated (HA) with limited knowledge regarding community-associated (CA) infections. Here, we investigated the molecular epidemiology of clinical isolates of CA-CDI and HA-CDI in a Swedish county. Data and isolates (n = 156) of CDI patients (n = 122) from Jönköping county, October 2017-March 2018, were collected and classified as CA (without previous hospital care or onset ≤2 days after admission or >12 weeks after discharge from hospital) or HA (onset >3 days after hospital admission or within 4 weeks after discharge). Molecular characterization of isolates included PCR ribotyping (n = 156 isolates) and whole genome sequencing with single nucleotide polymorphisms (SNP) analysis (n = 53 isolates). We classified 47 patients (39%) as CA-CDI and 75 (61%) as HA-CDI. Between CA-CDI and HA-CDI patients, we observed no statistically significant differences regarding gender, age, 30-day mortality or recurrence. Ribotype 005 (RR 3.1; 95% CI: 1.79-5.24) and 020 (RR 2.5; 95% CI: 1.31-4.63) were significantly associated with CA-CDI. SNP analysis identified seven clusters (0-2 SNP difference) involving 17/53 isolates of both CA-CDI and HA-CDI. Molecular epidemiology differed between CA-CDI and HA-CDI and WGS analysis suggests transmission of CDI within and between hospitals and communities.

Global spatial dynamics and vaccine-induced fitness changes of Bordetella pertussis.

As with other pathogens, competitive interactions between Bordetella pertussis strains drive infection risk. Vaccines are thought to perturb strain diversity through shifts in immune pressures; however, this has rarely been measured because of inadequate data and analytical tools. We used 3344 sequences from 23 countries to show that, on average, there are 28.1 transmission chains circulating within a subnational region, with the number of chains strongly associated with host population size. It took 5 to 10 years for B. pertussis to be homogeneously distributed throughout Europe, with the same time frame required for the United States. Increased fitness of pertactin-deficient strains after implementation of acellular vaccines, but reduced fitness otherwise, can explain long-term genotype dynamics. These findings highlight the role of vaccine policy in shifting local diversity of a pathogen that is responsible for 160,000 deaths annually.

Evaluation of 11 SARS-CoV-2 antibody tests by using samples from patients with defined IgG antibody titers.

We evaluated the performance of 11 SARS-CoV-2 antibody tests using a reference set of heat-inactivated samples from 278 unexposed persons and 258 COVID-19 patients, some of whom contributed serial samples. The reference set included samples with a variation in SARS-CoV-2 IgG antibody titers, as determined by an in-house immunofluorescence assay (IFA). The five evaluated rapid diagnostic tests had a specificity of 99.0% and a sensitivity that ranged from 56.3 to 81.6% and decreased with low IFA IgG titers. The specificity was > 99% for five out of six platform-based tests, and when assessed using samples collected ≥ 22 days after symptom onset, two assays had a sensitivity of > 96%. These two assays also detected samples with low IFA titers more frequently than the other assays. In conclusion, the evaluated antibody tests showed a heterogeneity in their performances and only a few tests performed well with samples having low IFA IgG titers, an important aspect for diagnostics and epidemiological investigations.

Impact of ribotype on Clostridioides difficile diagnostics.

This study investigates the performance of diagnostic methods for detection of Clostridioides difficile infection in Sweden, including impact of PCR ribotype on diagnostic performance. Between 2011 and 2016, a total of 17,878 stool samples from 26 laboratories were tested by either well-type enzyme immunoassays (EIAs), membrane bound EIAs, cell cytotoxicity neutralization assay (CTA), or nucleic acid amplification tests (NAATs) and subsequently cultured for C. difficile. Roughly half of the samples (9454/17878) were subjected to diagnostic testing both on the fecal sample and on the 1323 isolated C. difficile strains. All C. difficile isolates were typed by PCR ribotyping, and the isolates were classified as toxigenic or non-toxigenic based on the empirical knowledge of the association between toxin-positivity and ribotype. The overall sensitivity, specificity, and positive and negative predictive values were highest for NAATs and membrane EIAs. Ribotype-specific sensitivity varied greatly between methods and ribotypes. All methods had 100% sensitivity against ribotype 027 and 013. For other types, the sensitivity ranged from 33 to 85% in fecal samples and from 78 to 100% on isolates. For the most prevalent ribotypes (014, 020, and 001), the sensitivity varied between 38 and 100% in the fecal samples, with the lowest sensitivity observed for well-type EIAs and CTA. The large variation in diagnostic sensitivity implies that type distribution significantly affects the outcome when evaluating diagnostic performance. Furthermore, performing comparative studies of diagnostic tests in settings with high prevalence of ribotype 027 will overestimate the general performance of diagnostic tests.

National Surveillance for Clostridioides difficile Infection, Sweden, 2009-2016.

We report results from a national surveillance program for Clostridioides difficile infection (CDI) in Sweden, where CDI incidence decreased by 22% and the proportion of multidrug-resistant isolates decreased by 80% during 2012-2016. Variation in incidence between counties also diminished during this period, which might be attributable to implementation of nucleic acid amplification testing as the primary diagnostic tool for most laboratories. In contrast to other studies, our study did not indicate increased CDI incidence attributable the introduction of nucleic acid amplification testing. Our results also suggest that successful implementation of hygiene measures is the major cause of the observed incidence decrease. Despite substantial reductions in CDI incidence and prevalence of multidrug-resistant isolates, Sweden still has one of the highest CDI incidence levels in Europe. This finding is unexpected and warrants further investigation, given that Sweden has among the lowest levels of antimicrobial drug use.

Survey of Clostridium difficile infection surveillance systems in Europe, 2011.

To develop a European surveillance protocol for Clostridium difficile infection (CDI), existing national CDI surveillance systems were assessed in 2011. A web-based electronic form was provided for all national coordinators of the European CDI Surveillance Network (ECDIS-Net). Of 35 national coordinators approached, 33 from 31 European countries replied. Surveillance of CDI was in place in 14 of the 31 countries, comprising 18 different nationwide systems. Three of 14 countries with CDI surveillance used public health notification of cases as the route of reporting, and in another three, reporting was limited to public health notification of cases of severe CDI. The CDI definitions published by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) and the European Centre for Disease Prevention and Control (ECDC) were widely used, but there were differing definitions to distinguish between community- and healthcare-associated cases. All CDI surveillance systems except one reported annual national CDI rates (calculated as number of cases per patient-days). Only four surveillance systems regularly integrated microbiological data (typing and susceptibility testing results). Surveillance methods varied considerably between countries, which emphasises the need for a harmonised European protocol to allow consistent monitoring of the CDI epidemiology at European level. The results of this survey were used to develop a harmonised EU-wide hospital-based CDI surveillance protocol.

High Molecular Weight Typing with MALDI-TOF MS - A Novel Method for Rapid Typing of Clostridium difficile.

Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping. Among 500 isolates representing 59 PCR ribotypes a total of 35 high molecular weight types could be resolved. Although less discriminatory than PCR ribotyping, the method is extremely fast and simple, and supports for cost-effective screening of isolates during outbreak situations.

Comparison of strain typing results for Clostridium difficile isolates from North America.

Accurate strain typing is critical for understanding the changing epidemiology of Clostridium difficile infections. We typed 350 isolates of toxigenic C. difficile from 2008 to 2009 from seven laboratories in the United States and Canada. Typing was performed by PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and restriction endonuclease analysis (REA) of whole-cell DNA. The Cepheid Xpert C. difficile test for presumptive identification of 027/NAP1/BI isolates was also tested directly on original stool samples. Of 350 isolates, 244 (70%) were known PCR ribotypes, 224 (68%) were 1 of 8 common REA groups, and 187 (54%) were known PFGE types. Eighty-four isolates typed as 027, NAP1, and BI, and 83 of these were identified as presumptive 027/NAP1/BI by Xpert C. difficile. Eight additional isolates were called presumptive 027/NAP1/BI by Xpert C. difficile, of which three were ribotype 027. Five PCR ribotypes contained multiple REA groups, and three North American pulsed-field (NAP) profiles contained both multiple REA groups and PCR ribotypes. There was modest concordance of results among the three methods for C. difficile strains, including the J strain (ribotype 001 and PFGE NAP2), the toxin A-negative 017 strain (PFGE NAP9 and REA type CF), the 078 animal strain (PFGE NAP7 and REA type BK), and type 106 (PFGE NAP11 and REA type DH). PCR-ribotyping, REA, and PFGE provide different but overlapping patterns of strain clustering. Unlike the other methods, the Xpert C. difficile 027/NAP1/BI assay gave results directly from stool specimens, required only 45 min to complete, but was limited to detection of a single strain type.

Rapid and sensitive loop-mediated isothermal amplification test for Clostridium difficile detection challenges cytotoxin B cell test and culture as gold standard.

Compared to the composite gold standard cytotoxin B assay and toxigenic culture, the loop-mediated isothermal amplification (LAMP) test for Clostridium difficile had a sensitivity and specificity of 98%, positive predictive value of 92%, and negative predictive value of >99%. A one-hour turnaround time for the LAMP test provides rapid diagnosis and cost savings.

Impact of strain type on detection of toxigenic Clostridium difficile: comparison of molecular diagnostic and enzyme immunoassay approaches.

A multicenter clinical trial assessed the performance of the Cepheid Xpert C. difficile assay on stool specimens collected from patients suspected of having Clostridium difficile infection (CDI). A total of 2,296 unformed stool specimens, collected from seven study sites, were tested by Xpert C. difficile enrichment culture followed by cell culture cytotoxicity testing of the isolates (i.e., toxigenic culture with enrichment) and the study sites' standard C. difficile test methods. The methods included enzyme immunoassay (EIA), direct cytotoxin testing, and two- and three-step algorithms using glutamate dehydrogenase (GDH) screening followed by either EIA or EIA and an in-house PCR assay. All C. difficile strains were typed by PCR-ribotyping. Compared to results for toxigenic culture with enrichment, the sensitivity, specificity, and positive and negative predictive values of the Xpert assay were 93.5, 94.0, 73.0, and 98.8%, respectively. The overall sensitivity of the EIAs compared to that of enrichment culture was 60.0%, and the sensitivity of combined GDH algorithms was 72.9%; both were significantly lower than that of Xpert C. difficile (P < 0.001 and P = 0.03, respectively). The sensitivity of the EIA was significantly lower than that of the Xpert C. difficile assay for detection of ribotypes 002, 027, and 106 (P < 0.0001, P < 0.0001, and P = 0.004, respectively, Fisher's exact test), and the sensitivity of GDH algorithms for ribotypes other than 027 was lower than that for Xpert C. difficile (P < 0.001). The Xpert C. difficile assay is a simple, rapid, and accurate method for detection of toxigenic C. difficile in unformed stool specimens and is minimally affected by strain type compared to EIA and GDH-based methods.

Induction of toxins in Clostridium difficile is associated with dramatic changes of its metabolism.

Certain amino acids, and cysteine in particular, promptly blocked toxin expression in Clostridium difficile strain VPI 10463 when added to late-exponential-phase peptone-yeast cultures, i.e. prior to normal induction of toxins A and B. Glucose reduced toxin yields by 80-fold, but only when supplemented at inoculation. Forty upregulated C. difficile proteins were identified during maximum toxin expression, and most of these were enzymes involved in energy exchange, e.g. succinate, CO/folate and butyrate metabolism. Transcription of tcdA (toxin operon) and folD (CO/folate operon) was induced by 20- and 10-fold, respectively, and with strikingly similar kinetics between OD 0.8 and 1.2. The sigma factors tcdR and sigH were upregulated simultaneously with tcdA and folD (3.5-fold increase of mRNA level), whereas transcription of tcdC, codY, sigB and sigL showed little or no correlation with that of tcdA and folD. The results suggest a connection between toxin expression, alternative energy metabolism and initial sporulation events in C. difficile.

Increased sporulation rate of epidemic Clostridium difficile Type 027/NAP1.

Clostridium difficile PCR ribotype 027 comprised 0.2% of a collection of Swedish isolates in 1997-2001 (3 of 1,325 isolates). These isolates had lower moxifloxacin MICs than the epidemic type 027 isolates, but they had the same tcdC sequence and toxin yield. Type 027 produced 3- to 13-fold more toxin than did major Swedish types. One epidemic strain (027/NAP1a) sporulated more than did other type 027 isolates, a feature that should contribute to its survival and spread.

Frequent emergence of resistance in Clostridium difficile during treatment of C. difficile-associated diarrhea with fusidic acid.

Samples from patients with Clostridium difficile-associated diarrhea (CDAD) that were randomized to fusidic acid (n = 59) or metronidazole (n = 55) therapy for 7 days were cultured for Clostridium difficile in feces on days 1, 8 to 13, and 35 to 40. Of the patients who were culture positive only before treatment, 77% (36/47) were permanently cured (no treatment failure and no clinical recurrence), compared to 54% (22/41) of those with persistence of C. difficile at one or both follow-ups (P = 0.03). A similar association between bacterial persistence and a worse outcome of therapy was seen in both treatment groups. Resistance to fusidic acid was found in 1 of 88 pretherapy isolates available, plus in at least 1 subsequent isolate from 55% (11/20) of patients who remained culture-positive after fusidic acid therapy. In 10 of these 11 patients, the resistant follow-up isolate(s) belonged to the same PCR ribotype as the susceptible day 1 isolate, confirming frequent emergence of resistance to fusidic acid during treatment. Despite this, 5 of these 11 patients were permanently cured with fusidic acid, relative to 5 of 9 patients with susceptible C. difficile at follow-up (P = 1.0). None of the 36 PCR ribotypes of C. difficile identified was associated with any particular clinical outcome or emergence of fusidic acid resistance. In conclusion, culture positivity for C. difficile was common after both fusidic acid and metronidazole therapy and was associated with treatment failure or recurrence of CDAD. Development of resistance in C. difficile was frequent in patients given fusidic acid, but it was without apparent negative impact on therapeutic efficacy in the actual CDAD episode.

Antimicrobial susceptibility pattern of Clostridium difficile and its relation to PCR ribotypes in a Swedish university hospital.

All 238 Clostridium difficile isolates were susceptible to metronidazole and vancomycin, whereas 84% and 1% were resistant to clindamycin and fusidic acid. Etest MICs for metronidazole were lower than agar dilution MICs (P < 0.01) but without difference in susceptible-intermediate-resistant categorization. No particular PCR ribotype was associated with clindamycin or fusidic acid resistance.

Correlation of disease severity with fecal toxin levels in patients with Clostridium difficile-associated diarrhea and distribution of PCR ribotypes and toxin yields in vitro of corresponding isolates.

We investigated in vivo and in vitro yields of toxins A and B from and PCR ribotypes of Clostridium difficile isolates from 164 patients with differing severities of C. difficile-associated diarrhea (CDAD) (patients were grouped as follows: <3 loose stools per day, n = 45; 3 to 10 per day, n = 97; >10 per day, n = 22). The median fecal toxin levels in each group were 0.5, 6.8, and 149 U/g feces (P < 0.001), respectively. Patients with severe diarrhea also had more-frequent occurrence of blood in stool and vomiting, but there was no association with fecal toxin levels per se. There was no correlation between fecal toxin level and toxin yield in vitro for the corresponding C. difficile isolate or between its PCR ribotype and disease severity. A broad range of toxin yields among isolates belonging to major PCR ribotypes indicated a presence of many subtypes. We hypothesize that bacterial and host factors that affect C. difficile toxin levels in feces are important determinants of symptoms in CDAD patients. An inverse correlation between toxin yield and spore count (r = 0.66) in stationary-phase cultures supported the notion that toxin production and sporulation represent opposite alternative survival strategies for C. difficile cells facing nutrient shortage.

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions.

Data are presented from two-dimensional (2-D) PAGE analysis of Mycobacterium tuberculosis strain Harlingen grown during aerobic and anaerobic culture, according to a modified Wayne dormancy model. M. tuberculosis cultures were grown to the transition point between exponential growth and stationary phase in the presence of oxygen (7 days) and then part of the cultures was shifted to anaerobic conditions for 16 days. Growth declined similarly during aerobic and anaerobic conditions, whereas the ATP consumption rapidly decreased in the anaerobic cultures. 2-D PAGE revealed 50 protein spots that were either unique to, or more abundant during, anaerobic conditions and 16 of these were identified by MALDI-TOF. These proteins were the alpha-crystalline homologue (HspX), elongation factor Tu (Tuf), GroEL2, succinyl-CoA : 3-oxoacid-CoA transferase (ScoB), mycolic acid synthase (CmaA2), thioredoxin (TrxB2), beta-ketoacyl-ACP synthase (KasB), l-alanine dehydrogenase (Ald), Rv2005c, Rv2629, Rv0560c, Rv2185c and Rv3866. Some protein spots were found to be proteolytic fragments, e.g. HspX and GroEL2. These data suggest that M. tuberculosis induces expression of about 1 % of its genes in response to dormancy.

Epidemiology and molecular characterization of Clostridium difficile strains from patients with diarrhea: low disease incidence and evidence of limited cross-infection in a Swedish teaching hospital.

We prospectively studied the epidemiology of Clostridium difficile-associated diarrhea (CDAD) in a 900-bed hospital over the course of 12 months by PCR-ribotyping of C. difficile isolates. A total of 304 cases were diagnosed, corresponding to an overall incidence of 7/1,000 admissions, with higher rates in nephrology, hematology, and organ transplantation wards (37, 30, and 21/1,000), and 72% were classified as hospital associated (onset in hospital or onset at home but after a hospital stay within 2 months). All 382 isolates from 227 of 304 (75%) patients available for PCR-ribotyping were typeable, yielding 70 PCR-ribotypes. The three most common types comprised 30% of hospital-associated and 34% of community-associated cases, indicating import via admitted patients as a major source of C. difficile strains occurring in the hospital. Of the 227 patients studied, 38% each contributed 2 to 13 fecal samples positive for C. difficile over the course of the study period. Repeat isolates of the same PCR-ribotype as the first isolate were found in 79% of these patients and in 95% of specimens delivered within 30 days, compared to 63% of those obtained at 31 to 204 days. Nosocomial acquisition of CDAD, defined as the proportion of cases sharing C. difficile type and admitted to the same ward within 2 or 12 months, was 20% and 32% of hospital-associated cases and 14% and 23% of all cases, respectively. Thus, most CDAD cases diagnosed over the course of the study period, including those associated with hospitalization, appeared to be caused by endogenous C. difficile strains rather than by strains truly being acquired in the hospital.

Expression of Clostridium difficile toxins A and B and their sigma factor TcdD is controlled by temperature.

Growth temperature was found to control the expression of toxins A and B in Clostridium difficile VPI 10463, with a maximum at 37 degrees C and low levels at 22 and 42 degrees C in both peptone yeast (PY) and defined media. The up-regulation of toxin A and B mRNA and protein levels upon temperature upshift from 22 to 37 degrees C followed the same kinetics, showing that temperature control occurred at the level of transcription. Experiments with Clostridium perfringens using gusA as a reporter gene demonstrated that both toxin gene promoters were temperature controlled and that their high activity at 37 degrees C was dependent on the alternative sigma factor TcdD. Furthermore, tcdD was found to be autoinduced at 37 degrees C. Glucose down-regulated all these responses in the C. perfringens constructs, similar to its impact on toxin production in C. difficile PY broth cultures. C. difficile proteins induced at 37 degrees C and thus coregulated with the toxins by temperature were demonstrated by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as enzymes involved in butyric acid production and as electron carriers in oxidation-reduction reactions. The regulation of toxin production in C. difficile by temperature is a novel finding apparently reflecting an adaptation of the expression of its virulence to mammalian hosts.

Effects of the Min system on nucleoid segregation in Escherichia coli.

The Min system of Escherichia coli directs cell division to the mid-cell by a mechanism that involves the dynamic localization of all of its three constituent proteins, MinC, MinD and MinE. Both the Min system and the nucleoid regulate cell division negatively and strains of E. coli lacking a functional Min system can divide at nucleoid-free cell poles in addition to the nucleoid-free region between newly segregated nucleoids. Interestingly, E. coli strains with a defective Min system have disturbed nucleoid segregation and the cause for this disturbance is not known. It is reported here that growth conditions promoting a higher frequency of polar divisions also lead to a more pronounced disturbance in nucleoid segregation. In strains with an intact Min system, expression of MinE, but not of MinD, from an inducible promoter was followed by impaired nucleoid segregation. These results suggest that the disturbed nucleoid segregation in min mutants is not caused by polar divisions per se, nor by impaired resolution of chromosome dimers in min mutants, leaving open the possibility that the Min system has a direct effect on nucleoid segregation. It is also shown how the disturbed nucleoid segregation can explain in part the unexpected finding that the clear majority of cells in min mutant populations contain 2(n) (n=0, 1, 2.) origins of replication.

Proteins released during high toxin production in Clostridium difficile.

The mechanism by which toxins A and B are released by Clostridium difficile is unknown and information about the other extracellular proteins of this bacterium is limited. The authors identified exported proteins from C. difficile strain VPI 10463 during conditions promoting high toxin production. Toxins A and B were released in a 1:1 ratio and the proportion of toxin in the extracellular fraction reached 50% during the stationary phase as compared to a proportion of <1% for typical cytoplasmic proteins, showing that toxin export was not due to bacterial lysis. A 47 kDa protein, released with similar kinetics to the toxins, was processed and showed weak similarity to the channel-forming protein TolC. Another protein released during high toxin production was unprocessed and showed similarity to XkdK encoded by the prophage PBSX in Bacillus subtilis, a protein supposedly exported via phage-specific holins. The two most abundant extracellular C. difficile proteins, found during both high and low toxin production, were processed and identified as shed S-layer proteins. As shown by N-terminal sequencing and PCR-based methods, there was a considerable sequence variation of the S-layer gene slpA in different serogroup reference strains. To conclude, C. difficile uses the classical Sec-dependent and probably also holin-like pathways to secrete a comparatively small repertoire of proteins.