Multiple Ribotypes Research Articles

Phase variation of a signal transduction system controls Clostridioides difficile colony morphology, motility, and virulence.

Recent work has revealed that Clostridioides difficile, a major cause of nosocomial diarrheal disease, exhibits phenotypic heterogeneity within a clonal population as a result of phase variation. Many C. difficile strains representing multiple ribotypes develop two colony morphotypes, termed rough and smooth, but the biological implications of this phenomenon have not been explored. Here, we examine the molecular basis and physiological relevance of the distinct colony morphotypes produced by this bacterium. We show that C. difficile reversibly differentiates into rough and smooth colony morphologies and that bacteria derived from the isolates display discrete motility behaviors. We identified an atypical phase-variable signal transduction system consisting of a histidine kinase and two response regulators, named herein colony morphology regulators RST (CmrRST), which mediates the switch in colony morphology and motility behaviors. The CmrRST-regulated surface motility is independent of flagella and type IV pili, suggesting a novel mechanism of cell migration in C. difficile. Microscopic analysis of cell and colony structure indicates that CmrRST promotes the formation of elongated bacteria arranged in bundled chains, which may contribute to bacterial migration on surfaces. In a hamster model of acute C. difficile disease, the CmrRST system is required for disease development. Furthermore, we provide evidence that CmrRST phase varies during infection, suggesting that the intestinal environment impacts the proportion of CmrRST-expressing C. difficile. Our findings indicate that C. difficile employs phase variation of the CmrRST signal transduction system to generate phenotypic heterogeneity during infection, with concomitant effects on bacterial physiology and pathogenesis.

Evolutionary and Genomic Insights into Clostridioides difficile Sequence Type 11: a Diverse Zoonotic and Antimicrobial-Resistant Lineage of Global One Health Importance.

Clostridioides difficile (Clostridium difficile) sequence type 11 (ST11) is well established in production animal populations worldwide and contributes considerably to the global burden of C. difficile infection (CDI) in humans. Increasing evidence of shared ancestry and genetic overlap of PCR ribotype 078 (RT078), the most common ST11 sublineage, between human and animal populations suggests that CDI may be a zoonosis. We performed whole-genome sequencing (WGS) on a collection of 207 ST11 and closely related ST258 isolates of human and veterinary/environmental origin, comprising 16 RTs collected from Australia, Asia, Europe, and North America. Core genome single nucleotide variant (SNV) analysis identified multiple intraspecies and interspecies clonal groups (isolates separated by ≤2 core genome SNVs) in all the major RT sublineages: 078, 126, 127, 033, and 288. Clonal groups comprised isolates spread across different states, countries, and continents, indicative of reciprocal long-range dissemination and possible zoonotic/anthroponotic transmission. Antimicrobial resistance genotypes and phenotypes varied across host species, geographic regions, and RTs and included macrolide/lincosamide resistance (Tn6194 [ermB]), tetracycline resistance (Tn6190 [tetM] and Tn6164 [tet44]), and fluoroquinolone resistance (gyrA/B mutations), as well as numerous aminoglycoside resistance cassettes. The population was defined by a large "open" pan-genome (10,378 genes), a remarkably small core genome of 2,058 genes (only 19.8% of the gene pool), and an accessory genome containing a large and diverse collection of important prophages of the Siphoviridae and Myoviridae This study provides novel insights into strain relatedness and genetic variability of C. difficile ST11, a lineage of global One Health importance.IMPORTANCE Historically, Clostridioides difficile (Clostridium difficile) has been associated with life-threatening diarrhea in hospitalized patients. Increasing rates of C. difficile infection (CDI) in the community suggest exposure to C. difficile reservoirs outside the hospital, including animals, the environment, or food. C. difficile sequence type 11 (ST11) is known to infect/colonize livestock worldwide and comprises multiple ribotypes, many of which cause disease in humans, suggesting CDI may be a zoonosis. Using high-resolution genomics, we investigated the evolution and zoonotic potential of ST11 and a new closely related ST258 lineage sourced from diverse origins. We found multiple intra- and interspecies clonal transmission events in all ribotype sublineages. Clones were spread across multiple continents, often without any health care association, indicative of zoonotic/anthroponotic long-range dissemination in the community. ST11 possesses a massive pan-genome and numerous clinically important antimicrobial resistance elements and prophages, which likely contribute to the success of this globally disseminated lineage of One Health importance.

The ClosER study: results from a three-year pan-European longitudinal surveillance of antibiotic resistance among prevalent Clostridium difficile ribotypes, 2011–2014

ObjectivesUntil the introduction of fidaxomicin, antimicrobial treatment for Clostridium difficile infection (CDI) was limited to metronidazole and vancomycin. The changing epidemiology of CDI and the emergence of epidemic C. difficile PCR ribotype 027 necessitate continued surveillance to identify shifts in antibiotic susceptibility. ClosER, currently the largest pan-European epidemiological study of C. difficile ribotype distribution and antibiotic susceptibility, aimed to undertake antimicrobial resistance surveillance pre- and post-introduction of fidaxomicin. MethodsBetween July 2011 and July 2014, 39 sites across 22 European countries submitted 2830 C. difficile isolates for ribotyping, toxin testing and susceptibility testing to metronidazole, vancomycin, fidaxomicin, rifampicin, moxifloxacin, clindamycin, imipenem, chloramphenicol and tigecycline. ResultsRibotypes 027, 014, 001, 078, 020, 002, 126, 015 and 005 were most frequently isolated, and emergent ribotypes 198 and 356 were identified in Hungary and Italy, respectively. All isolates were susceptible to fidaxomicin, with scarce resistance to metronidazole (0.2%, 6/2694), vancomycin (0.1%, 2/2694) and tigecycline (0%). Rifampicin, moxifloxacin and clindamycin resistance was evident in multiple ribotypes. Lack of ribotype diversity correlated with greater antimicrobial resistance. Epidemic ribotypes (027/001) were associated with multiple antimicrobial resistance, and ribotypes 017, 018 and 356 with high-level resistance. Additional factors may also influence local ribotype prevalence. ConclusionsFidaxomicin susceptibility was retained post-introduction, and resistance to metronidazole and vancomycin was rare. Continued surveillance is needed, with more accurate classification and clarification of ribotype subtypes to further understand their role in the spread of resistance. Other factors may also influence changes in prevalence of C. difficile ribotypes with reduced antibiotic susceptibility.

Pan-European longitudinal surveillance of antibiotic resistance among prevalent Clostridium difficile ribotypes

Clostridium difficile infection remains a major healthcare burden. Until the recent introduction of fidaxomicin, antimicrobial treatments were limited to metronidazole and vancomycin. The emergence of epidemic C. difficile PCR ribotype 027 and its potential link to decreased antibiotic susceptibility highlight the lack of large-scale antimicrobial susceptibility and epidemiological data available. We report results of epidemiological and antimicrobial susceptibility investigations of C. difficile isolates collected prior to fidaxomicin introduction, establishing important baseline data. Thirty-nine sites in 22 countries submitted a total of 953 C. difficile isolates for PCR ribotyping, toxin testing, and susceptibility testing to metronidazole, vancomycin, fidaxomicin, rifampicin, moxifloxacin, clindamycin, imipenem, chloramphenicol, and tigecycline. Ninety-nine known ribotypes were identified. Ribotypes 027, 014, 001/072, and 078 were most frequently isolated in line with previous European studies. There was no evidence of resistance to fidaxomicin, and reduced susceptibility to metronidazole and vancomycin was also scarce. Rifampicin, moxifloxacin, and clindamycin resistance (13%, 40%, and 50% of total isolates, respectively) were evident in multiple ribotypes. There was a significant correlation between lack of ribotype diversity and greater antimicrobial resistance (measured by cumulative resistance score). Well-known epidemic ribotypes 027 and 001/072 were associated with multiple antimicrobial resistance, but high levels of resistance were also observed, particularly in 018 and closely related emergent ribotype 356 in Italy. This raises the possibility of antimicrobial exposure as the underlying reason for their appearance, and highlights the need for ongoing epidemiological and antimicrobial resistance surveillance.

Effect of ribotype on all-cause mortality following Clostridium difficile infection

Clostridium difficile infection (CDI) is significantly associated with subsequent all-cause mortality. Although a number of studies have investigated mortality associated with CDI, few have compared all-cause mortality between ribotypes. We aimed to estimate all-cause mortality following CDI and to investigate the relationship between mortality, ribotype and other available variables. We undertook a retrospective cohort study. All patients with toxin-positive CDI in North East England between July 2009 and June 2011 were matched to death registration data. Differences in all-cause 30-day case fatality were explored using Poisson regression with robust error variances. For survival analysis, an accelerated failure time model with generalized gamma distribution was chosen. In total, 1426 patients were included. All-cause case fatality was 10.2%, 16.4%, 25.7% and 38.1% at 7, 14, 30 and 90 days respectively. In multivariate analysis, ribotype 027 (risk ratio: 1.34; 95% confidence interval: 1.02-1.75) and ribotype 015 (0.46; 0.26-0.82) were significantly associated with higher and lower all-cause 30-day case fatality rates, respectively. In survival analysis, only ribotype 015 had significantly lower predicted mortality (P=0.008). Patients whose infection was hospital-acquired had significantly higher predicted mortality (P<0.001). This is the first population-based study of comparative mortality between multiple ribotypes. Our study identified a high rate of all-cause mortality following CDI. We found evidence of variability in mortality between ribotypes in this cohort with mortality significantly higher for ribotype 027 at 30 days following diagnosis and significantly lower for ribotype 015.

Disruption of the Human Gut Microbiota following Norovirus Infection

The gut microbiota, the collection of all bacterial members in the intestinal tract, plays a key role in health. Disruption of the indigenous microbiota by a variety of stressors, including antibiotic therapy and intestinal infections, is associated with multiple health problems. We sought to determine if infection with Norovirus disrupts the gut microbiota. Barcoded pyrosequencing of the 16S rRNA-encoding gene was used to characterize the stool microbiota in Norovirus-infected human patients (n = 38). While the microbiota in most infected patients (n = 31) resembled that seen in uninfected healthy controls, a minority of patients (n = 7) possessed a significantly altered microbiota characterized by reduced relative numbers of Bacteriodetes and a corresponding increase in Proteobacteria. In these patients, the increase in Proteobacteria was due to a single operational taxonomic unit (OTU) of Escherichia coli. We cultured E. coli from Norovirus-infected patients and characterized them using PCR-ribotyping and virulence factor analysis. Multiple ribotypes were encountered, but none possessed typical virulence factors commonly carried by enteropathogenic E. coli strains. Microbiota disruption and elevated Proteobacteria were not significantly correlated to patient age, gender, sampling time following illness onset, or overall gut inflammation. These results demonstrate that some patients have a disrupted microbiota following Norovirus infection, and therefore may be at elevated risk for long-term health complications.

Characterization of Vibrio cholerae O139 belonging to multiple ribotypes and isolated from diarrhoeal patients in Kerala, southern India

Twenty-four Vibrio cholerae O139 strains isolated from Kerala, southern India were characterized by PCR, CTX typing and ribotyping; all of which, except three strains, carried the core of the CTX genetic element, colonization-toxin co-regulated pilus, the adherence outer membrane protein, haemolysin, central regulatory protein encoded toxR, SXT genetic element, and produced cholera toxin and biofilm. Results of RFLP analysis revealed twenty-one of the O139 strains possess two copies of CTXΦ and pre-CTXФ always preceded by tandemly arranged RS1 element; one had two copies of pre-CTXΦ and two a single copy of pre-CTXΦ. Nucleotide sequencing detected classical ctxB in CTXETΦ and CTXCalcФ with additional change at 28th amino acid position of CTXCalcФ. Ribotype analysis revealed the presence of multiple ribotypes, including B-I and B-II, and new ribotypes designated B-VIIIa, B-VIIIb and B-IX, not reported earlier among V. cholerae O139 strains. These observations thus indicate that genetic recombination or mutations had occurred in conserved rrn operon and variations in CTXΦ may have implications on the evolution of the organism.

Use of 16S rRNA and rpoB Genes as Molecular Markers for Microbial Ecology Studies

Several characteristics of the 16S rRNA gene, such as its essential function, ubiquity, and evolutionary properties, have allowed it to become the most commonly used molecular marker in microbial ecology. However, one fact that has been overlooked is that multiple copies of this gene are often present in a given bacterium. These intragenomic copies can differ in sequence, leading to identification of multiple ribotypes for a single organism. To evaluate the impact of such intragenomic heterogeneity on the performance of the 16S rRNA gene as a molecular marker, we compared its phylogenetic and evolutionary characteristics to those of the single-copy gene rpoB. Full-length gene sequences and gene fragments commonly used for denaturing gradient gel electrophoresis were compared at various taxonomic levels. Heterogeneity found between intragenomic 16S rRNA gene copies was concentrated in specific regions of rRNA secondary structure. Such "heterogeneity hot spots" occurred within all gene fragments commonly used in molecular microbial ecology. This intragenomic heterogeneity influenced 16S rRNA gene tree topology, phylogenetic resolution, and operational taxonomic unit estimates at the species level or below. rpoB provided comparable phylogenetic resolution to that of the 16S rRNA gene at all taxonomic levels, except between closely related organisms (species and subspecies levels), for which it provided better resolution. This is particularly relevant in the context of a growing number of studies focusing on subspecies diversity, in which single-copy protein-encoding genes such as rpoB could complement the information provided by the 16S rRNA gene.

Escherichia coli Pathogenicity Island-Like Domains

In their molecular epidemiological analysis of Escherichia coli urosepsis isolates, Bingen-Bidois et al. interpret the finding of a pathogenicity island (PAI) IIJ96-like domain in multiple ribotypes as conflicting with previous reports, which ostensibly describe this element as being confined to a single clone (1). This is potentially misleading, since the two references cited actually pertain to papG allele I, which is a hallmark of a different PAI from that of strain J96, i.e., PAI I. Indeed, papG allele I, which was absent from the population analyzed by Bingen-Bidois et al. (rather than being found in multiple ribotypes), is quite rare, having been found almost exclusively in strains closely related to strain J96 (4). In contrast, many previous reports have documented the co-occurrence of papG allele III, hly, and cnf1 (which implies the presence of a PAI IIJ96-like domain, as discussed by Bingen-Bidois et al.) in multiple group B2-derived lineages (3). These include clonal groups 1 and 2 of Cherifi et al. (2) (which equate, respectively, electrophoretic types [ETs] 1 and 21 of Whittam et al. [7] and ETs 47 and 42 of Maslow et al. [6], the latter clonal group including archetypal strain 536), the O2:K5/K7:H1 clonal group (ET 39 of Maslow et al.), and the O18:K1:H7 clonal group that includes archetypal strains RS218 (meningitis) and NU14 (cystitis) (3, 5).

Serotyping and Ribotyping of Salmonella Using Restriction Enzyme PvuII

The subtyping and identification of bacterial pathogens throughout food processing and production chains is useful to the new hazard analysis critical control point–based food safety plans. Traditional manual serotyping remains the primary means of subtyping Salmonella isolates. Molecular biology techniques, however, offer the promise of more rapid and sensitive subtyping of Salmonella. This study evaluates the potential of restriction enzyme PvuII, followed by probing with the rRNA operon from Escherichia coli, to generate serotype-specific DNA fingerprints. A total of 32 identified serotypes were found with an overall agreement in 208 of the 259 (80%) isolates tested between U.S. Department of Agriculture serotype identification and riboprint serotype identification. Many of the isolates that did not correlate were serotype identified as Salmonella Montevideo, which indicates that for this serotype, there are multiple ribotypes. When Salmonella Montevideo isolates were not included, the ribotype identification agreed with serotyping in 207 of the 231 (90%) isolates. The primary outcome of any ribotyping procedure is to give distinct ribotype patterns. This extensive poultry epidemiological study demonstrates that, in addition to ribotype patterns, the identification of isolates to known serotypes provides the investigator with additional information that can be more useful than traditional epidemiology and isolate identification studies.

Comparative genetic characterization of Listeria monocytogenes isolates from human and animal listeriosis cases.

Listeria monocytogenes isolates from human sporadic and epidemic cases (n=119) and from animal cases (n=76) were characterized by automated ribotyping and PCR-restriction fragment length polymorphism (PCR-RFLP) typing of the virulence genes actA and hly. This combination of typing methods differentiated 39 distinctive strains, each reflecting a unique combination of ribotypes, hly and actA alleles. Simpson's index of discrimination indicated a high discriminatory ability of ribotyping for both animal (0.867) and human isolates (0.857), which was further increased by the addition of hly and actA typing (0.916 and 0.904, respectively). Ribotype and hly allele data were further used to group isolates into three genetically distinct lineages. Each lineage is composed of several ribotype fragment subsets, each of which contains multiple ribotypes characterized by common ribotype fragments. To determine whether certain clones of L. monocytogenes show indications for unique pathogenic potential or host specificity, frequency distributions for five genetic characteristics (i.e. lineage, ribotype, ribotype fragment subset and hly and actA allele) were calculated for isolates from animal cases, human epidemic cases and human sporadic cases. Lineage III isolates were found less frequently in human cases (1 of 119 isolates) than in animal cases (8 of 76 isolates; P=0.003). These results suggest the possibility of host specificity for non-primate mammals among lineage III strains. In addition, lineage I strains were found more frequently among human cases than among animal cases (P<0.001). Among the eight hly alleles observed, hly allele 1 was more common among human isolates as compared to animal isolates (P=0.002). We also identified one ribotype (DUP-1030) which was significantly more common among animal isolates (P=0.005) and one ribotype (DUP-1038; lineage I) which was significantly more common among human epidemic isolates as compared to human sporadic isolates (P<0.001). These findings confirm the presence of clonal groups of L. monocytogenes, which appear to be characterized by unique virulence or host specificity patterns. This study also establishes baseline data describing the genetic diversity of human and animal L. monocytogenes isolates which can be utilized in future surveillance programmes to track the emergence of new strains.

Ribotype diversity of porcine Pasteurella multocida from Australia.

To use the technique of ribotyping to investigate the genetic diversity of Australian isolates of Pasteurella multocida associated with outbreaks of clinical disease in Australian pigs. One hundred and seven porcine P multocida isolates were analysed by ribotyping using the restriction enzymes HpaII and HindIII. The genetic population structure of the Australian porcine P multocida isolates was determined through statistical analysis of the joint ribotype patterns, and this was then compared with biochemical and epidemiological data available for the population. A total of 25 combined ribotypes were recognised, which were grouped into five ribotype clusters. Despite the deliberate selection of diverse isolates, the study revealed only a limited degree of genetic diversity. Fourteen of the ribotypes contained multiple isolates, and 12 of these ribotypes were present on more than one farm. Three of the seven biovars analysed in the study showed very limited diversity. All fifteen biovar 2 isolates (subsp multocida) were found in a single cluster (III), while all four biovar 8 isolates, which correspond to P multocida subsp gallicida, were allocated by themselves to a single cluster (IV). All nine of the biovar 12 isolates (lactose-positive subsp multocida) were assigned to a single cluster (I), together with the single biovar 14 isolate, which was the only other lactose-positive isolate in the population (ODC-negative). A limited number of ribotypes of P multocida are associated with Australian pigs. The majority of these ribotypes are widely distributed across multiple farms, and across multiple states. Individual farms can possess multiple ribotypes of P multocida. Some of the unusual biochemical variants of P multocida present in Australian pigs have a very limited genetic diversity. The nature of pig production in Australia, primarily involving continuous flow systems with few closed herds, has possibly contributed to the widespread distribution of a limited number ribotypes.

Outbreak of encephalitic listeriosis in Holstein bulls

Five Holstein bulls in a herd of 478 bulls were diagnosed with clinical encephalitic listeriosis. Four bulls survived after intensive therapy with oxytetracycline, dexamethasone, and oral fluid with electrolytes. The bull which did not survive had histological findings of lymphoplasmacytic encephalitis and inflammation of the trigeminal ganglion, which were consistent with the diagnosis of clinical listeriosis. A suspect silage sample was submitted for laboratory evaluation. The silage had a pH of 6.9 and was positive for Listeria monocytogenes. In vitro antibiotic testing of the organism isolated revealed resistance to penicillin, the drug commonly recommended for the treatment of listeriosis. It is concluded that caution should be exercised, however, when evaluating in vitro susceptibility results associated with a listeriosis outbreak because multiple ribotypes may be involved.

Diversity of Streptococcus agalactiae and Staphylococcus aureus ribotypes recovered from New York dairy herds

Abstract Objectives To develop a reference database for characterization of bovine Staphylococcus aureus and Streptococcus agalactiae strains by automated ribotyping and to use it to assess the discriminatory power of this typing procedure and the geographic distribution of Sta aureus and Str agalactiae strains in New York state dairy herds. Sample Population 22 commercial dairy herds. Procedure Isolates of Sta aureus and Str agalactiae from bovine milk were identified by standard bacteriologic procedures, then typed by automated ribotyping. Antimicrobial susceptibility of isolates was tested in vitro. Two indicators made from the data were percentage of farms with multiple ribotypes and percentage of single ribotypes found in several geographic regions. Standard bacteriologic diagnosis, automated ribotyping, and determination of antibiograms (Kirby-Bauer method) also were done. Results Of 50 Sta aureus and 44 Str agalactiae isolates from composite milk samples of 12 and 10 herds, respectively, 18 and 14 ribotypes, respectively, were identified. The discriminatory power of automated ribotyping was approximately 0.96 (Hunter-Gaston's formula). A higher percentage of herds with Sta aureus had multiple ribotypes. The most common Sta aureus ribotypes tended to have broader geographic distribution. Some Sta aureus ribotypes were significantly associated with antibiotic resistance profiles. Conclusions Automated ribotyping appears to characterize bovine strains of bacteria associated with intramammary infections with a high discriminatory index. Potential applications include identification of strains that appear to have broad geographic distribution suggesting interfarm transfer, discrimination between recurrent versus new intramammary infections (ie, for control of Str agalactiae and Sta aureus), and evaluation of antibiotic therapy. (Am J Vet Res 1997;58:482–487)

Carriage of multiple ribotypes of non-encapsulated Haemophilus influenzae in aboriginal infants with otitis media.

Ribotyping with the restriction enzyme XbaI was used to study the dynamics of Carriage of non-encapsulated Haemophilus influenzae (NCHi) in Aboriginal infants at risk of otitis media. Carriage rates of NCHi in the infants in the community were very high; the median age for detection was 50 days and colonization was virtually 100% by 120 days of age and persisted at a high level throughout the first year of life [1]. Eighteen different ribotypes of NCHi were identified from 34 positive swabs taken from 3 infants over a period of 9 months. The same ribotypes were recovered for up to 3 months from consecutive swabs of individual infants, and 12 of 27 swabs (44.4%) yielded two ribotypes from four colonies typed. Statistical analysis suggested that most swabs would have been positive for two ribotypes if enough colonies had been typed although the second most frequent ribotype was detected on average in only 13% of strains. Early colonization and carriage of multiple ribotypes of NCHi may help to explain the chronicity of carriage and thus the persistence of otitis media in Aboriginal infants.

Molecular Fingerprinting ofPasteurella MultocidaAssociated with Progressive Atrophic Rhinitis in Swine Herds

Ninety-six nasal isolates of Pasteurella multocida from swine herds with progressive atrophic rhinitis were characterized by restriction endonuclease analysis (REA) of whole-cell DNA, ribotyping, and plasmid analysis. For REA, bacterial DNA was digested with SmaI and electrophoresed in 0.7% agarose, and fragments were visualized with UV light. For ribotyping, EcoRI-digested and electrophoresed restriction fragments of whole-cell DNA were transferred to nitrocellulose membranes, hybridized with gamma-32P-labeled Escherichia coli ribosomal RNA, and visualized by autoradiography. Phenotypes of isolates were toxigenic capsular type D (n = 51), nontoxigenic type D (n = 28), nontoxigenic type A (n = 16), and toxigenic type A (n = 1). Plasmids of various sizes were evident in 92.2% and 17.9% of toxigenic and nontoxigenic D strains, respectively, but were absent from all type A strains. Among the 4 phenotypes, there were 17 REA profiles and 6 ribotypes. For 3 of 17 REA patterns, multiple ribotypes were evident, and several REA types were evident in 5 of 6 ribotypes. Thirty-seven isolates of toxigenic capsular type D from Australian herds were either SmaI type B or C and ribotype 2, whereas 14 toxigenic D isolates from the USA and other countries were more heterogeneous (7 REA types and 6 ribotypes). The fingerprinting results provided evidence in support of the hypothesis of a single source infection in Australia associated with the introduction of breeding pigs from overseas.