Difficile Clinical Isolates Research Articles

Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates.

To describe, for the first time (to the best of our knowledge), the genetic mechanisms of vancomycin resistance in clinical isolates of Clostridioides difficile ribotype 027. Clinical isolates and laboratory mutants were analysed: genomically to identify resistance mutations; by transcriptional analysis of vanGCd, the vancomycin resistance operon encoding lipid II d-alanine-d-serine that is less bound by vancomycin than native lipid II d-alanine-d-alanine; by imaging of vancomycin binding to cell walls; and for changes in vancomycin bactericidal activity and autolysis. Vancomycin-resistant laboratory mutants and clinical isolates acquired mutations to the vanSR two-component system that regulates vanGCd. The substitutions impaired VanSR's function, resulting in constitutive transcription of vanGCd. Resistance was reversed by silencing vanG, encoding d-alanine-d-serine ligase in the vanGCd operon. In resistant cells, vancomycin was less bound to the cell wall septum, the site where vancomycin interacts with lipid II. Vancomycin's bactericidal activity was reduced against clinical isolates and laboratory mutants (64 and ≥1024 mg/L, respectively) compared with WT strains (4 mg/L). Truncation of the potassium transporter TrkA occurred in laboratory mutants, which were refractory to autolysis, accounting for their survival in high drug concentrations. Ribotype 027 evolved first-step resistance to vancomycin by constitutively expressing vanGCd, which is otherwise silent. Experimental evolutions and bactericidal assays show that ribotype 027 can acquire mutations to drastically enhance its tolerance to vancomycin. Thus, further epidemiological studies are warranted to examine the extent to which vancomycin resistance impacts clinical outcomes and the potential for these strains to evolve higher-level resistance, which would be devastating.

Curcumin: A natural derivative with antibacterial activity against Clostridium difficile

ObjectivesThe rapid emergence of hypervirulent Clostridium difficile (C. difficile) isolates and the paucity of effective anti-clostridial antibiotics call for extensive research to identify new treatment options. This study aimed to test the anti-clostridial activity of bioactive extracts of turmeric, which is a natural herb widely known for its profound medicinal properties. MethodsThe MICs of turmeric derivatives were determined against 27 C. difficile strains, including hypervirulent (BI/NAP1/027) and clinical toxigenic isolates. Additionally, their ability to inhibit C. difficile toxin production and spore formation was investigated. Furthermore, the safety profiles of turmeric derivatives regarding their effects on human gut microflora – such as Bacteroides, Lactobacillus and Bifidobacterium – were evaluated. ResultsCurcuminoids, the major phytoconstituents of turmeric – including curcumin, demethoxycurcumin and bisdemethoxycurcumin – inhibited growth of C. difficile at concentrations ranging from 4 to 32μg/mL. Additionally, curcuminoids showed no negative effect on major populating species of the human gut. Curcumin was more effective than fidaxomicin in inhibiting C. difficile toxin production, but less so in inhibiting spore formation. ConclusionThe findings suggest that curcumin has potential as an anti-clostridial agent. More work is needed to further investigate the efficacy of curcumin as a stand-alone drug or as a supplement of current drugs of choice, as it has no antagonistic activities but might overcome their drawbacks.

Integrated genomic epidemiology and phenotypic profiling of Clostridium difficile across intra-hospital and community populations in Colombia

Clostridium difficile, the causal agent of antibiotic-associated diarrhea, has a complex epidemiology poorly studied in Latin America. We performed a robust genomic and phenotypic profiling of 53 C. difficile clinical isolates established from diarrheal samples from either intrahospital (IH) or community (CO) populations in central Colombia. In vitro tests were conducted to evaluate the cytopathic effect, the minimum inhibitory concentration of ten antimicrobial agents, the sporulation efficiency and the colony forming ability. Eleven different sequence types (STs) were found, the majority present individually in each sample, however in three samples two different STs were isolated. Interestingly, CO patients were infected with STs associated with hypervirulent strains (ST-1 in Clade-2). Three coexistence events (two STs simultaneously detected in the same sample) were observed always involving ST-8 from Clade-1. A total of 2,502 genes were present in 99% of the isolates with 95% of identity or more, it represents a core genome of 28.6% of the 8,735 total genes identified in the set of genomes. A high cytopathic effect was observed for the isolates positive for the two main toxins but negative for binary toxin (TcdA+/TcdB+/CDT− toxin production type), found only in Clade-1. Molecular markers conferring resistance to fluoroquinolones (cdeA and gyrA) and to sulfonamides (folP) were the most frequent in the analyzed genomes. In addition, 15 other markers were found mostly in Clade-2 isolates. These results highlight the regional differences that C. difficile isolates display, being in this case the CO isolates the ones having a greater number of accessory genes and virulence-associated factors.

Antimicrobial susceptibility and mechanisms of resistance of Greek Clostridium difficile clinical isolates.

This study examined the antimicrobial susceptibility and resistance mechanisms of Clostridium difficile recovered in Greek hospitals during 2012-2015. C. difficile isolates (n=88) were collected from clinically-confirmed C. difficile infection from symptomatic patients in 10 Greek hospitals. Minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined by Etest. Isolates were typed by multilocus sequence typing (MLST). Toxin and resistance genes were detected by PCR. Chromosomal mutations in gyrA, gyrB and rpoB were identified by PCR and sequencing. The genetic environment of resistance genes was characterised by Illumina sequencing. The 88 C. difficile isolates comprised 27 sequence types (STs), with ST37 (n=26) and ST11 (n=21) being the most prevalent. All isolates were susceptible to vancomycin and metronidazole, with variable resistance rates to other antimicrobials. Of the 88 isolates, 45.5% were multidrug-resistant and the majority belonged to ST11 and ST37. The presence of chromosomal mutations in gyrA, gyrB and rpoB was mainly observed in high-risk clones such as ST11 and ST37. The antimicrobial resistance genes ermB, mefA, msrA and tetM were identified at different prevalences and combinations. Additionally, cfrB and cfrC were identified for the first time in Greece and were carried by a Tn6218 transposon and a novel plasmid, respectively. To our knowledge, this is the first study examining the resistance profiles and respective mechanisms of C. difficile recovered in Greek hospitals. Gut commensals such as C. difficile may serve as hubs for further transfer of antimicrobial resistance genes.

Correlation between restriction endonuclease analysis and PCR ribotyping for the identification of Clostridioides (Clostridium) difficile clinical strains

Restriction endonuclease analysis (REA) and PCR ribotyping are two typing systems that have been frequently utilized for molecular epidemiologic characterization of Clostridioides (Clostridium) difficile. To correlate typing data obtained from each method, we performed both REA and PCR ribotyping on a large and diverse set of historical and contemporary C. difficile infection clinical isolates. Eighty isolates were selected from each reference laboratory in the United States (Microbiology Reference Laboratory, Hines VA Medical Center) and United Kingdom (Clostridium difficile Network for England and Northern Ireland laboratory, University of Leeds). The 160 isolates were assigned to 82 unique ribotypes and 51 unique REA groups (116 unique REA types). In general, concordance between typing methods was good. Dendrogram analysis of PCR ribotype band patterns demonstrated close genetic relationships among strain types with discordant REA and ribotype assignments. While REA typing was more discriminatory, several REA types in this study were further discriminated by PCR ribotyping, indicating that discriminatory value of these typing methods may be strain dependent. These data will assist with molecular epidemiologic surveillance of strains identified by these two commonly used C. difficile typing systems.

Identification and Characterization of Clostridium difficile Sequence Type 37 Genotype by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Clostridium difficile multilocus sequence type 37 (ST37), which mainly corresponds to ribotype 017, has been a dominant genotype circulating in China. In this study, we report the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to analyze and characterize 204 C. difficile clinical isolates, including 49 ST37 and 155 non-ST37 isolates collected in China and other countries. The distributions of two major protein peaks (m/z 3,242 and 3,286) were significantly different between ST37 and non-ST37 prototype strains and clinical isolates. This difference was reproducible when analysis was performed on different colonies in different runs. This finding was repeated and confirmed by both bioMérieux Vitek MS and Bruker Microflex LT systems on isolates recovered from a variety of geographic regions worldwide. The combination of the two peaks was present in 47 of 49 ST37 isolates, resulting in a sensitivity of 95.9%. In contrast, the peak combination was absent in 153 of 155 non-ST37 isolates, resulting in a specificity of 98.7%. Our results suggest that MALDI-TOF MS is a rapid and reliable tool to identify C. difficile genotype ST37. Work is in progress to characterize the two molecules having peaks at m/z 3,242 and 3,286, which appear to be specific to C. difficile genotype ST37.

In Vitro and In Vivo Activities of DS-2969b, a Novel GyrB Inhibitor, against Clostridium difficile.

DS-2969b is a novel GyrB inhibitor that is currently under clinical development for the treatment of Clostridium difficile infection (CDI). In this study, the in vitro and in vivo activities of DS-2969b were evaluated. DS-2969b inhibited the supercoiling activity of C. difficile DNA gyrase. DS-2969b showed potent in vitro activity against C. difficile clinical isolates with a MIC90 of 0.06 μg/ml, which was 2-, 32-, and 16-fold lower than the MIC90s of fidaxomicin, vancomycin, and metronidazole, respectively. DS-2969b did not select spontaneously resistant mutants of various C. difficile strains at 4× MIC, and the frequency of resistance development was less than 4.8 × 10-9 In a hamster CDI model, 5-day oral administration of DS-2969b conferred complete protection from recurrence and mortality at 0.3 mg/kg of body weight once a day, in contrast to a 50% survival rate with fidaxomicin at 3 mg/kg once a day and 0% with vancomycin at a 50-mg/kg/dose twice a day. Even a single oral administration of 1 mg/kg of DS-2969b in the CDI model exhibited 100% animal survival without recurrence. DS-2969b was also efficacious by 5-day subcutaneous administration in the CDI model. DS-2969b showed similar levels of fecal excretion after intravenous and oral administrations in rats. These data support further development of DS-2969b as a drug for oral and intravenous treatment of CDI.

Antibiotic Resistances of Clostridium difficile.

The rapid evolution of antibiotic resistance in Clostridium difficile and the consequent effects on prevention and treatment of C. difficile infections (CDIs) are matter of concern for public health. Antibiotic resistance plays an important role in driving C. difficile epidemiology. Emergence of new types is often associated with the emergence of new resistances and most of epidemic C. difficile clinical isolates is currently resistant to multiple antibiotics. In particular, it is to worth to note the recent identification of strains with reduced susceptibility to the first-line antibiotics for CDI treatment and/or for relapsing infections. Antibiotic resistance in C. difficile has a multifactorial nature. Acquisition of genetic elements and alterations of the antibiotic target sites, as well as other factors, such as variations in the metabolic pathways and biofilm production, contribute to the survival of this pathogen in the presence of antibiotics. Different transfer mechanisms facilitate the spread of mobile elements among C. difficile strains and between C. difficile and other species. Furthermore, recent data indicate that both genetic elements and alterations in the antibiotic targets can be maintained in C. difficile regardless of the burden imposed on fitness, and therefore resistances may persist in C. difficile population in absence of antibiotic selective pressure.

Surveillance of antibiotic resistance among common Clostridium difficile ribotypes in Hong Kong

Incidence of Clostridium difficile infection (CDI) is rapidly increasing and it poses a major health burden globally. However, data regarding the epidemiology of CDI in Asia are limited. We aimed to characterize the antimicrobial susceptibility patterns of common ribotypes of toxigenic C. difficile in Hong Kong. Fifty-three PCR ribotypes were identified among 284 toxigenic C. difficile clinical isolates. The five most prevalent ribotypes were 002 (13%), 017 (12%), 014 (10%), 012 (9.2%), and 020 (9.5%). All tested C. difficile strains remained susceptible to metronidazole, vancomycin, meropenem and piperacillin/tazobactam, but highly resistant to cephalosporins. Of the fluoroquinolones, highest resistance to ciprofloxacin was observed (99%), followed by levofloxacin (43%) and moxifloxacin (23%). The two newly emerged PCR ribotypes, 017 and 002, demonstrated high levels of co-resistance towards clindamycin, tetracycline, erythromycin and moxifloxacin. PCR ribotypes 017 and 002 with multi-drug resistance are rapidly emerging and continuous surveillance is important to monitor the epidemiology of C. difficile to prevent outbreaks of CDI.

Revisiting the Role of Csp Family Proteins in Regulating Clostridium difficile Spore Germination.

Clostridium difficile causes considerable health care-associated gastrointestinal disease that is transmitted by its metabolically dormant spore form. Upon entering the gut, C. difficile spores germinate and outgrow to produce vegetative cells that release disease-causing toxins. C. difficile spore germination depends on the Csp family of (pseudo)proteases and the cortex hydrolase SleC. The CspC pseudoprotease functions as a bile salt germinant receptor that activates the protease CspB, which in turn proteolytically activates the SleC zymogen. Active SleC degrades the protective cortex layer, allowing spores to outgrow and resume metabolism. We previously showed that the CspA pseudoprotease domain, which is initially produced as a fusion to CspB, controls the incorporation of the CspC germinant receptor in mature spores. However, study of the individual Csp proteins has been complicated by the polar effects of TargeTron-based gene disruption on the cspBA-cspC operon. To overcome these limitations, we have used pyrE-based allelic exchange to create individual deletions of the regions encoding CspB, CspA, CspBA, and CspC in strain 630Δerm Our results indicate that stable CspA levels in sporulating cells depend on CspB and confirm that CspA maximizes CspC incorporation into spores. Interestingly, we observed that csp and sleC mutants spontaneously germinate more frequently in 630Δerm than equivalent mutants in the JIR8094 and UK1 strain backgrounds. Analyses of this phenomenon suggest that only a subpopulation of C. difficile 630Δerm spores can spontaneously germinate, in contrast with Bacillus subtilis spores. We also show that C. difficile clinical isolates that encode truncated CspBA variants have sequencing errors that actually produce full-length CspBA.IMPORTANCEClostridium difficile is a leading cause of health care-associated infections. Initiation of C. difficile infection depends on spore germination, a process controlled by Csp family (pseudo)proteases. The CspC pseudoprotease is a germinant receptor that senses bile salts and activates the CspB protease, which activates a hydrolase required for germination. Previous work implicated the CspA pseudoprotease in controlling CspC incorporation into spores but relied on plasmid-based overexpression. Here we have used allelic exchange to study the functions of CspB and CspA. We determined that CspA production and/or stability depends on CspB and confirmed that CspA maximizes CspC incorporation into spores. Our data also suggest that a subpopulation of C. difficile spores spontaneously germinates in the absence of bile salt germinants and/or Csp proteins.

Antimicrobial activity of fidaxomicin against Clostridium difficile clinical isolates in Aichi area in Japan.

We evaluated the susceptibility of 100 Japanese Clostridium difficile isolates to fidaxomicin, a new macrocyclic antibiotic. The minimum inhibitory concentration (MIC) range of fidaxomicin was 0.03-0.5μg/mL, with a MIC for inhibition of 50% (MIC50) of 0.12μg/mL, and for inhibition of 90% (MIC90) of 0.25μg/mL. We also evaluated the susceptibilities of the same 100 C.difficile isolates to vancomycin, metronidazole, moxifloxacin, clindamycin, meropenem, and ampicillin. Of all the antibiotics tested, fidaxomicin showed the most potent antimicrobial activity against this group of C.difficile isolates. MIC levels against C.difficile isolates, including those producing binary toxin, did not substantially differ from those previously reported in Europe, North America and Taiwan.

Ribotypes associated with Clostridium difficile outbreaks in Brazil display distinct surface protein profiles

Clostridium difficile is a spore-forming anaerobic intestinal pathogen that causes Clostridium difficile infection (CDI). C. difficile is the leading cause of toxin-mediated nosocomial antibiotic-associated diarrhea. The pathogenesis of CDI is attributed to two major virulence factors, TcdA and TcdB toxins, that cause the symptomatic infection. C. difficile also expresses a number of key proteins, including cell wall proteins (CWPs). S-layer proteins (SLPs) are CWPs that form a paracrystalline surface array that coats the surface of the bacterium. SLPs have a role in C. difficile binding to the gastrointestinal tract, but their importance in virulence need to be better elucidated. Here, we describe bottom-up proteomics analysis of surface-enriched proteins fractions obtained through glycine extraction of five C. difficile clinical isolates from Brazil using gel-based and gel-free approaches. We were able to identify approximately 250 proteins for each strain, among them SlpA, Cwp2, Cwp6, CwpV and Cwp84. Identified CWPs presented different amino acid coverage, which might suggest differences in post-translational modifications. Proteomic analysis of SLPs from ribotype 133, agent of C. difficile outbreaks in Brazil, revealed unique proteins and provided additional information towards in depth characterization of the strains causing CDI in Brazil.

Surveillance of Antibiotic Resistance among Hospital- and Community-Acquired Toxigenic Clostridium difficile Isolates over 5-Year Period in Kuwait.

Clostridium difficile infection (CDI) is a leading and an important cause of diarrhea in a healthcare setting especially in industrialized countries. Community-associated CDI appears to add to the burden on healthcare setting problems. The aim of the study was to investigate the antimicrobial resistance of healthcare-associated and community-acquired C. difficile infection over 5 years (2008–2012) in Kuwait. A total of 111 hospital-acquired (HA-CD) and 35 community-acquired Clostridium difficile (CA-CD) clinical isolates from stool of patients with diarrhoea were studied. Antimicrobial susceptibility testing of 15 antimicrobial agents against these pathogens was performed using E test method. There was no evidence of resistance to amoxicillin-clavulanic acid, daptomycin, linezolid, piperacillin-tazobactam, teicoplanin and vancomycin by both HA-CD and CA-CD isolates. Metronidazole had excellent activity against CA-CD but there was a 2.9% resistance rate against HA-CD isolates. Ampicillin, clindamycin, levofloxacin and imipenem resistance rates among the HC-CD vs. CA-CD isolates were 100 vs. 47.4%; 43 vs. 47.4%; 100 vs. 100% and 100 vs. 89%, respectively. An unexpected high rifampicin resistance rate of 15.7% emerged amongst the HA-CD isolates. In conclusion, vancomycin resistance amongst the HA-CD and CA-CD isolates was not encountered in this series but few metronidazole resistant hospital isolates were isolated. High resistance rates of ampicillin, clindamycin, levofloxacin, and imipenem resistance were evident among both CA-CD and HA-CD isolates. Rifampicin resistance is emerging among the HA-CD isolates.

Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile.

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding $3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell-wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose-dependent manner. We achieved a more than 4-log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. Biotechnol. Bioeng. 2016;113: 2568-2576. © 2016 Wiley Periodicals, Inc.

Molecular and epidemiologic study of Clostridium difficile reveals unusual heterogeneity in clinical strains circulating in different regions in Portugal

Clostridium difficile infection (CDI) represents a great healthcare burden in developed countries. The emergence of the epidemic PCR ribotype (RT) 027 and its acquired fluoroquinolones resistance have accentuated the need for an active surveillance of CDI. Here we report the first countrywide study of CDI in Portugal with the characterization of 498 C. difficile clinical isolates from 20 hospitals in four regions in Portugal regarding RT, virulence factors and antimicrobial susceptibility. We identified 96 RTs with marked variations between and within regions, as only six RTs appeared in all four regions. RT027 was the most frequent RT overall (18.5%) and among healthcare facility-associated isolates (19.6%), while RT014 was the most common among community-associated isolates (12%). The north showed a high RT diversity among isolates and a low moxifloxacin (MXF) resistance rate (11.9%), being the only region in which RT027 was not predominant. In contrast, the isolates from the centre presented the highest RT027 frequency, and 53.4% were resistant to MXF. Overall, MXF resistance (33.2%) was associated (p <0.001) with the presence of binary toxin genes and mutations in tcdC regardless of the RT. Both traits appeared in almost 30% of the strains. RT027 showed a reduced susceptibility to metronidazole (p <0.01), and RT126 had higher minimum inhibitory concentrations to vancomycin (p = 0.03) compared to other RTs. The present study highlights an unusual heterogeneity of RTs in Portugal, with a high frequency of hypervirulent RTs and the emergence of virulence factors in non-027 RTs, emphasizing the need for a surveillance system for CDI in Portugal.

Molecular characterization and antimicrobial susceptibility of tcdA-negative Clostridium difficile isolates from Guangzhou, China

This study aimed to investigate the molecular characteristics and antimicrobial susceptibility of Clostridium difficile clinical isolates in Guangzhou, China. One hundred twenty isolates were collected from Guangzhou General Hospital at the Guangzhou Military Command in China from March 2014 to April 2015, and 9 isolates were identified as tcdA-negative/tcdB-positive (A−B+) strains. Results showed that all of the strains were confirmed to be ST37 and 0 single nucleotide variants (SNVs) were found in the PaLoc region, and >60 SNVs were identified throughout the whole genome sequence. The results show the diversity of the antibiotic and gene mutations present in these strains. All of the A−B+ isolates were highly resistant to clindamycin and erythromycin; showed an average sensitivity to fluoroquinolones; and maintained a high susceptibility to metronidazole, vancomycin, and tigecycline.

Diagnostic testing for Clostridium difficile in Italian microbiological laboratories

A laboratory diagnosis survey of Clostridium difficile infection (CDI) was performed in Italy in 2012–2013. Questionnaires from 278 healthcare settings from 15 regions of Italy were collected and analysed. Eighty seven percent of the laboratories declared to routinely perform CDI diagnosis, 99% of them only after the clinician's request. Among the 216 laboratories providing information on the size of the hospitals in which they were located, 65 had more than 500 beds (large hospitals), while 151 had less than 500 beds (small hospitals). The average percentage of positive tests for C. difficile toxins was 12.2%. Almost half of the laboratories (42%) used immunoenzymatic assay (EIA) for Tox A/B as a stand-alone method, while only 34% used an algorithm for CDI as indicated by the European guidelines. A low percentage of laboratories performed molecular assays or C. difficile culture, 25% and 29%, respectively. Most laboratories (161/278) declared to type C. difficile strains, the majority in collaboration with a reference laboratory. Among the 103 C. difficile clinical isolates collected during the study, 31 different PCR-ribotypes were identified. PCR-ribotype 356/607 (27%) was predominant, followed by 018 (12%). These two PCR-ribotypes show 87.5% of similarity in ribotyping profile. PCR-ribotypes 027 and 078 represented 8% and 4% of the strains, respectively. Four PCR-ribotypes (027, 033, 078 and 126) were positive for the binary toxin CDT. In particular, PCR-ribotype 033 produces only CDT, and it has recently been associated with symptomatic cases. The majority of strains were multidrug resistant. In particular, all strains PCR-ribotypes 356/607 and 018 were resistant to moxifloxacin, rifampicin, erythromycin and clindamycin. The results obtained highlight the need to raise awareness to the microbiological diagnosis of CDI among clinicians and to implement and harmonize diagnostic methods for CDI in Italian laboratories in the perspective of a future national surveillance.

Doxycycline and Tigecycline: Two Friendly Drugs with a Low Association with Clostridium Difficile Infection.

Clostridium difficile infection (CDI) is known to be associated with prior exposure to many classes of antibiotics. Standard therapy for CDI (i.e., metronidazole and vancomycin) is associated with high recurrence rates. Although tetracycline derivatives such as tetracycline, doxycycline or tigecycline are not the standard therapeutic choices for CDI, they may serve as an alternative or a component of combination therapy. Previous tetracycline or doxycycline usage had been shown to have less association with CDI development. Tigecycline, a broad-spectrum glycylcycline with potency against many gram-positive or gram-negative pathogens, had been successfully used to treat severe or refractory CDI. The in vitro susceptibility of C. difficile clinical isolates to tigecycline in many studies showed low minimal inhibitory concentrations. Tigecycline can suppress in vitro toxin production in both historical and hypervirulent C. difficile strains and reduce spore production in a dose-dependent manner. Tetracycline compounds such as doxycycline, minocycline, and tigecycline possess anti-inflammatory properties that are independent of their antibiotic activity and may contribute to their therapeutic effect for CDI. Although clinical data are limited, doxycycline is less likely to induce CDI, and tigecycline can be considered one of the therapeutic choices for severe or refractory CDI.

Molecular characterization and antimicrobial susceptibilities of Clostridium difficile clinical isolates from Victoria, Australia

Some Australian strain types of Clostridium difficile appear unique, highlighting the global diversity of this bacterium. We examined recent and historic local isolates, finding predominantly toxinotype 0 strains, but also toxinotypes V and VIII. All isolates tested were susceptible to vancomycin and metronidazole, while moxifloxacin resistance was only detected in recent strains.

Variation in germination of Clostridium difficile clinical isolates correlates to disease severity

Over the past two decades, Clostridium difficile infections have been increasing in both number and severity throughout the world. As with other spore forming bacteria, germination is a vital step in the life cycle of this pathogen. Studies have examined differences in sporulation and toxin production among a number of C. difficile clinical isolates; however, few have examined differences in germination and the relationship between this phenotype and disease severity. Here, over 100 C. difficile isolates from the University of Michigan Health System were examined for overall germination in response to various combinations of known germinants (taurocholate) and co-germinants (glycine and histidine). Significant variation was observed among isolates under all conditions tested. Isolates representing ribotype 014-020, which was the most frequently isolated ribotype at our hospital, exhibited increased germination in the presence of taurocholate and glycine when compared to isolates representing other ribotypes. Interestingly, isolates that caused severe disease exhibited significantly lower germination in response to minimal germination conditions (taurocholate only), indicating increased control over germination in these isolates. These data provide a broad picture of C. difficile isolate germination and indicate a role for precise control of germination in disease severity.