SUMMARY Clostridium difficile infection (CDI) is the leading cause of antimicrobial and health care-associated diarrhea in humans, presenting a significant burden to global health care systems. In the last 2 decades, PCR- and sequence-based techniques, particularly whole-genome sequencing (WGS), have significantly furthered our knowledge of the genetic diversity, evolution, epidemiology, and pathogenicity of this once enigmatic pathogen. C. difficile is taxonomically distinct from many other well-known clostridia, with a diverse population structure comprising hundreds of strain types spread across at least 6 phylogenetic clades. The C. difficile species is defined by a large diverse pangenome with extreme levels of evolutionary plasticity that has been shaped over long time periods by gene flux and recombination, often between divergent lineages. These evolutionary events are in response to environmental and anthropogenic activities and have led to the rapid emergence and worldwide dissemination of virulent clonal lineages. Moreover, genome analysis of large clinically relevant data sets has improved our understanding of CDI outbreaks, transmission, and recurrence. The epidemiology of CDI has changed dramatically over the last 15 years, and CDI may have a foodborne or zoonotic etiology. The WGS era promises to continue to redefine our view of this significant pathogen.

Erysipelothrix rhusiopathiae is a facultative, non-spore-forming, non-acid-fast, small, Gram-positive bacillus. The organism was first established as a human pathogen late in the nineteenth century. Three forms of human disease have been recognised since then. These include a localised cutaneous lesion form, erysipeloid, a generalised cutaneous form and a septicaemic form often associated with endocarditis. The organism is ubiquitous and able to persist for a long period of time in the environment, including marine locations. It is a pathogen or a commensal in a wide variety of wild and domestic animals, birds and fish. Swine erysipelas caused by E. rhusiopathiae is the disease of greatest prevalence and economic importance. Diseases in other animals include erysipelas of farmed turkeys, chickens, ducks and emus, and polyarthritis in sheep and lambs. Infection due to E. rhusiopathiae in humans is occupationally related, principally occurring as a result of contact with contaminated animals, their products or wastes, or soil. Erysipeloid is the most common form of infections in humans. While it has been suggested that the incidence of human infection could be declining due to technological advances in animal industries, infection still occurs in specific environments. Additionally, infection by the organism is possibly under-diagnosed due to the resemblance it bears to other infections, and problems encountered in isolation and identification. Various virulence factors have been suggested as being involved in the pathogenicity of E. rhusiopathiae. The presence of a hyaluronidase and neuraminidase has been recognised, and it was shown that neuraminidase plays a significant role in bacterial attachment and subsequent invasion into host cells. The role of hyaluronidase in the disease process is controversial. The presence of a heat labile capsule has been reported as important in virulence. Control of animal disease by sound husbandry, herd management, good sanitation and immunization procedures is recommended.

Increasing reports of antimicrobial resistance and limited new antibiotic discoveries and development have fuelled innovation in other research fields and led to a revitalization of bacteriophage (phage) studies in the Western world. Phage therapy mainly utilizes obligately lytic phages to kill their respective bacterial hosts, while leaving human cells intact and reducing the broader impact on commensal bacteria that often results from antibiotic use. Phage therapy is rapidly evolving and has resulted in cases of life-saving therapeutic use and multiple clinical trials. However, one of the biggest challenges this antibiotic alternative faces relates to regulations and policy surrounding clinical use and implementation beyond compassionate cases. This review discusses the multi-drug resistant Gram-negative pathogens of highest critical priority and summarizes the current state-of-the-art in phage therapy targeting these organisms. It also examines phage therapy in humans in general and the approaches different countries have taken to introduce it into clinical practice and policy. We aim to highlight the rapidly advancing field of phage therapy and the challenges that lie ahead as the world shifts away from complete reliance on antibiotics.

Escherichia coli from 138 fecal samples from aboriginal children, in whom no other enteric pathogen was isolated (including enterovirulent E. coli), were examined for HEp-2 cell adhesion. Twenty-five (36.8%) of 68 children with diarrhea and 32 (45.7%) of 70 without diarrhea had diffusely adherent isolates, which were thus not associated with diarrhea (P > .25). However, after age stratification, children > or = 18 months showed a significant association of diffusely adherent E. coli with diarrhea (P < or = .05). Enteroaggregative E. coli were isolated from 12 children with diarrhea (17.6%) and 15 without diarrhea (21.4%); thus, there was no association with diarrhea (P > or = .5). Sixteen children with diarrhea (23.5%) and 6 without diarrhea (8.6%) carried isolates that caused detachment of the HEp-2 cell monolayer from the glass coverslip when examined in the adhesion assay and were significantly associated with diarrhea (P < or = .05). These isolates, termed cell-detaching E. coli, were different from all recognized classes of enterovirulent E. coli.

The complete genome sequence of Clostridium difficile phage wC2 and comparisons to wCD119 and inducible prophages of CD630 The complete genomic sequence of a previously characterized temperate phage of Clostridium difficile, wC2, is reported. The genome is 56 538 bp and organized into 84 putative ORFs in six functional modules. The head and tail structural proteins showed similarities to that of C. difficile phage wCD119 and Streptococcus pneumoniae phage EJ-1, respectively. Homologues of structural and replication proteins were found in prophages 1 and 2 of the sequenced C. difficile CD630 genome. A putative holin appears unique to the C. difficile phages and was functional when expressed in Escherichia coli. Nucleotide sequence comparisons of wC2 to wCD119 and the CD630 prophage sequences showed relatedness between wC2 and the prophages, but less so to wCD119. wC2 integrated into a gene encoding a putative transcriptional regulator of the gntR family. wC2, wCD119 and CD630 prophage 1 genomes had a Cdu1-attP-integrase arrangement, suggesting that the pathogenicity locus (PaLoc) of C. difficile, flanked by cdu1, has phage origins. The attP sequences of wC2, wCD119 and CD630 prophages were dissimilar. wC2-related sequences were found in 84 % of 37 clinical C. difficile isolates and typed reference strains. INTRODUCTIONClostridium difficile has emerged as an important intestinal pathogen since the 1970s, continuing to plague hospital settings worldwide and causing recent epidemics in the USA and Canada (Loo et al., 2005;Warny et al., 2005). The major virulence factors of C. difficile are toxins A and B encoded by tcdA and tcdB respectively, which are located on a 19 kb genomic region termed the pathogenicity locus (PaLoc) (Braun et al., 1996;Hammond & Johnson, 1995). The toxins are positively regulated by TcdR (Mani & Dupuy, 2001;Rupnik et al., 2005) and are negatively regulated by TcdC (Hundsberger et al., 1997;Matamouros et al., 2006); they are encoded by tcdR and tcdC respectively, also on the PaLoc. Another toxin-associated gene, tcdE, appears phage related but its function is unknown (Tan et al., 2001). C. difficile acquires antibiotic resistance and virulence genes through plasmids and transposons (Bruggemann, 2005) shown to significantly contribute to genome plasticity (Sebaihia et al., 2006). It is possible that phages also contribute to variance in virulence-associated genes (Lemee et al., 2005) and to the emergence of outbreak strains . However, the prevalence of phage genes within C. difficile genomes is not known. In comparison to phages of Escherichia coli, Staphylococcus aureus and Lactobacillus species, the study of clostridial phages is in its infancy. Only one phage specific for C. difficile, temperate phage wCD119, has been sequenced , while two putative prophage sequences were detected in the recently sequenced genome of C. difficile CD630 (Sebaihia et al., 2006).

SUMMARY, or group B streptococcus (GBS), is a major neonatal pathogen. Recent data have elucidated the global prevalence of maternal and neonatal colonization, but gaps still remain in the epidemiology of this species. A number of phenotypic and genotypic classifications can be used to identify the diversity of GBS strains, and some are more discriminatory than others. This review explores the main schemes used for GBS epidemiology and further details the targets for epidemiological surveillance. Current screening practices across the world provide a unique opportunity to gain detailed information on maternal colonizing strains and neonatal disease-causing strains, which is vital for monitoring and therapeutics, if sufficient detail can be extracted. Deciphering which isolates are circulating within specific populations and recording targets within invasive strains are crucial steps in monitoring the implementation of therapeutics, such as vaccines, as well as developing novel therapies against prevalent GBS strains. Having a detailed understanding of global GBS epidemiology will prove invaluable for understanding the pathogenesis of this organism and equipping future prevention strategies for success.

The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages C2, C5, C6, and C8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of C2 showed nucleotide homology to the sequenced C. difficile strain CD630.Clostridium difficile has risen from relative obscurity 20 years ago to be an important hospital pathogen recognized as the cause of a wide spectrum of enteric diseases, including C. difficile-associated diarrhea (CDAD) (28). Almost all antimicrobials have now been implicated as being able to incite CDAD, including the two agents most commonly used for treatment, vancomycin and metronidazole (12). Thus, alternative nonantibiotic treatment modalities have been sought. There has been renewed interest in phage therapy (35) with the recent success of whole-phage and purified-phage components used in treating infections (7) or as antimicrobial agents (18,31). Although phages of the Clostridium species have been described previously (22), there have been a limited number of studies of C. difficile phages (20,27). Some studies deal with their use in strain typing (10, 32), but extensive studies of the molecular biology of C. difficile phages are lacking. The aim of this study, therefore, was to isolate and characterize bacteriophages specific for C. difficile as a preliminary step to assessing their potential as novel therapeutic agents.Fifty-six C. difficile strains were isolated from patients with CDAD in Sir Charles Gairdner Hospital, Perth, Western Australia, Australia, by previously described methods (29). Clostridium perfringens (13 isolates), Clostridium septicum (2 isolates), Staphylococcus aureus (10 isolates), Lactobacillus spp. (10 isolates), and Bacillus subtilis ATCC 6633 were obtained from the culture collection of The Western Australian Centre for Pathology and Medical Research. All cultures were incubated in an anaerobic chamber (80% N 2 , 10% CO 2 , and 10% H 2 ; model Mark III; Don Whitley Scientific Limited) at 37°C, except B. subtilis ATCC 6633. Stationary-phase (16-to 18-h) and log-phase (2.5-to 3-h) cultures of bacteria were prepared in brain heart infusion broth. MRS medium was also used for Lactobacillus spp. Anaerobic plaque assays were done on anaerobe basal agar (ABA) and overlay agar (0.74% [wt/vol] ABA, 0.01 M CaCl 2 , 0.4 M MgCl 2 ). Rogosa agar was also used for Lactobacillus spp., and aerobic plaque assays of S. aureus and B. subtilis were done on nutrient agar. C. difficile phages were not detected in environmental samples. Eight samples of soil and animal feces were collected from areas around and away from Sir Charles Gairdner Hospital; untreated sewage was collected from a treatment plant on five different occasions. Soil and fecal samples were mixed with 2 volumes of phosphate-buffered saline; these and the sewage samples were centrifuged at 17,700 ϫ g for 60 min at 4°C and filtered through 0.22-m-pore-size filters...

We determined the susceptibilities of 144 clinical and 49 environmental Aeromonas strains representing 10 different species to 26 antimicrobial agents by the agar dilution method. No single species had a predominantly nonsusceptible phenotype. A multidrug nonsusceptible pattern was observed in three (2.1%) clinical strains and two (4.0%) strains recovered from diseased fish. Common clinical strains were more resistant than the corresponding environmental isolates, suggesting that resistance mechanisms may be acquired by environmental strains from clinical strains.