Unveiling genome plasticity and a novel phage in Mycoplasma felis: Genomic investigations of four feline isolates.

Mycoplasma felis has been isolated from diseased cats and horses, but to date only a single fully assembled genome of this species, of an isolate from a horse, has been characterized. This study aimed to characterize and compare the completely assembled genomes of four clinical isolates of M. felis from three domestic cats, assembled with the aid of short- and long-read sequencing methods. The completed genomes encoded a median of 759 ORFs (range 743-777) and had a median average nucleotide identity of 98.2 % with the genome of the available equid origin reference strain. Comparative genomic analysis revealed the occurrence of multiple horizontal gene transfer events and significant genome reassortment. This had resulted in the acquisition or loss of numerous genes within the Australian felid isolate genomes, encoding putative proteins involved in DNA transfer, metabolism, DNA replication, host cell interaction and restriction modification systems. Additionally, a novel mycoplasma phage was detected in one Australian felid M. felis isolate by genomic analysis and visualized using cryo-transmission electron microscopy. This study has highlighted the complex genomic dynamics in different host environments. Furthermore, the sequences obtained in this work will enable the development of new diagnostic tools, and identification of future infection control and treatment options for the respiratory disease complex in cats.

Pasteurella multocida ST20 is widespread in Australian poultry farms and may infect wild waterbirds.

The bacterial agent that causes fowl cholera, Pasteurella multocida, was isolated from two deceased wild waterbirds in Victoria, Australia, in 2013. Whole genome sequence analysis placed the isolates into ST20, a subtype described in farmed chickens from Queensland, Australia and more recently in feedlot cattle and in pigs across a broader area of the continent. This study also found ST20 between 2009 and 2022 on three chicken farms and two turkey farms located in four Australian states. The sequences of 25 of these ST20 isolates were compared to 280 P. multocida genomes from 23 countries and to 94 ST20 Illumina datasets from Queensland that have been deposited in public databases. The ST20 isolates formed a single phylogenetic clade and were clustered into four sub-groups with highly similar genomes, possessing either LPS type 1 or type 3 loci. Various repertoires of mobile genetic elements were present in isolates from farmed, but not wild birds, suggesting complex histories of spill-over between avian populations and gene acquisition within farm environments. No major antimicrobial resistance was predicted in any of the ST20 isolates by the genomic analysis. The closest relative of these isolates was a ST394 bovine respiratory tract isolate from Queensland, which differed from ST20 by only one allele and carried beta-lactam and tetracycline resistance genes. These findings underline the importance of understanding the role of wild and commercial birds in the maintenance of fowl cholera, and of implementing regular epidemiological surveillance and biosecurity management programmes in wildlife, as well as free-range poultry farms.

Detection and Differentiation of Entomopathogenic Serratia spp. to Inform Reintroduction of the Critically Endangered Lord Howe Island Stick Insect Dryococelus australis.

Once rodents have been successfully eradicated from Lord Howe Island, Australia, the critically endangered Lord Howe Island stick insect (Dryococelus australis (Montrouzier)) may be reintroduced, a century after it was thought to have become extinct. In captive populations of D. australis, elevated mortalities have been associated with bacterial pathogens. To better define the infectious risk posed by entomopathogens to the reintroduction program, we investigated the bacteria isolated from captive D. australis kept at Melbourne Zoo and on Lord Howe Island and from environmental samples and free-living invertebrates collected on various parts of the island. At Melbourne Zoo, Serratia and Pseudomonas spp. were the bacteria most frequently isolated between 2013 and 2019. Serratia spp. were also the organisms most frequently isolated from insects sampled in April 2019 from the captive population on Lord Howe Island. In addition, Serratia spp. were isolated from a range of environmental samples collected on Lord Howe Island during March-April 2019. These environmental isolates had a broader range of biochemical and molecular characteristics than those obtained from the captive insect populations. A large proportion of these isolates were urease positive and had biochemical profiles previously not described for Serratia spp. This study highlights the need for better surveillance for potential pathogens in understudied regions and sites. We conclude that infections caused by Serratia spp. might pose a problem to the captive breeding program for D. australis but that the risk of introducing novel pathogens to Lord Howe Island through infected insects is low. Our study explores some of the potential risks involved in captive breeding and provides a valuable example of using pathogen surveillance to better inform an invertebrate conservation program.

Genomic characterization of Mycoplasma edwardii isolated from a dog bite induced cat wound reveals multiple horizontal gene transfer events and loss of the CRISPR/Cas system.

A domestic short hair cat (Felis catus) suffering from a purulent wound infection resulting from a dog bite was sampled for bacterial culture and isolation as the wound had been unresponsive to prolonged antimicrobial treatment. A mycoplasma was isolated from the wound. Whole genome sequencing of the isolate was performed using short-read Illumina and long-read Oxford Nanopore chemistry, and the organism was identified as Mycoplasma edwardii. Comparison of the genome sequence of the isolate to a reference M. edwardii genome sequence (canid isolate) identified the loss of several key bacterial factors involved in genome editing, as well the insertion of several novel ORFs most closely related to those found in other canine mycoplasmas, specifically Mycoplasma canis, M. cynos, M. molare and M. maculosa. This is only the second known report of disease caused by M. edwardii in a non-canid species, and the first report of it infecting and causing clinical disease in a cat.

Whole Genome Sequencing of Avian Pathogenic Escherichia coli Causing Bacterial Chondronecrosis and Osteomyelitis in Australian Poultry.

Bacterial chondronecrosis with osteomyelitis (BCO) impacts animal welfare and productivity in the poultry industry worldwide, yet it has an understudied pathogenesis. While Avian Pathogenic Escherichia coli (APEC) are known to be one of the main causes, there is a lack of whole genome sequence data, with only a few BCO-associated APEC (APECBCO) genomes available in public databases. In this study, we conducted an analysis of 205 APECBCO genome sequences to generate new baseline phylogenomic knowledge regarding the diversity of E. coli sequence types and the presence of virulence associated genes (VAGs). Our findings revealed the following: (i) APECBCO are phylogenetically and genotypically similar to APEC that cause colibacillosis (APECcolibac), with globally disseminated APEC sequence types ST117, ST57, ST69, and ST95 being predominate; (ii) APECBCO are frequent carriers of ColV-like plasmids that carry a similar set of VAGs as those found in APECcolibac. Additionally, we performed genomic comparisons, including a genome-wide association study, with a complementary collection of geotemporally-matched genomes of APEC from multiple cases of colibacillosis (APECcolibac). Our genome-wide association study found no evidence of novel virulence loci unique to APECBCO. Overall, our data indicate that APECBCO and APECcolibac are not distinct subpopulations of APEC. Our publication of these genomes substantially increases the available collection of APECBCO genomes and provides insights for the management and treatment strategies of lameness in poultry.

Global Phylogeny and F Virulence Plasmid Carriage in Pandemic Escherichia coli ST1193.

Lower urinary tract, renal, and bloodstream infections caused by phylogroup B2 extraintestinal pathogenic Escherichia coli (ExPEC) are a leading cause of morbidity and mortality. ST1193 is a phylogroup B2, multidrug-resistant sequence type that has risen to prominence globally, but a comprehensive analysis of the F virulence plasmids it carries is lacking. We performed a phylogenomic analysis of ST1193 (n = 707) whole-genome sequences from EnteroBase using entries with comprehensive isolation metadata. The data set comprised isolates from humans (n = 634 [90%]), including 339 (48%) from extraintestinal infection sites, and isolates from companion animals, wastewater, and wildlife. Phylogenetic analyses combined with gene detection and genotyping resolved an ST1193 clade structure segregated by serotype and F plasmid carriage. Most F plasmids fell into one of three related plasmid subtypes: F-:A1:B10 (n = 444 [65.97%]), F-:A1:B1 (n = 84 [12.48%]), and F-:A1:B20 (n = 80 [11.89%]), all of which carry the virulence genes cjrABC colocalized with senB (cjrABC-senB), a trademark signature of F29:A-:B10 subtype plasmids (pUTI89). To examine the phylogenetic relationship of these plasmids with pUTI89, complete sequences of F-:A1:B1 and F-:1:B20 plasmids were resolved. Unlike pUTI89, the most dominant and widely disseminated F plasmid that carries cjrABC-senB, F plasmids in ST1193 often carry a complex resistance region with an integron truncation (intI1Δ745) signature embedded within a structure assembled by IS26. Plasmid analysis shows that ST1193 has F plasmids that carry cjrABC-senB and ARG-encoding genes but lack tra regions and are likely derivatives of pUTI89. Further epidemiological investigation of ST1193 should seek to confirm its presence in human-associated environments and identify any potential agricultural links, which are currently lacking. IMPORTANCE We have generated an updated ST1193 phylogeny using publicly available sequences, reinforcing previous assertions that Escherichia coli ST1193 is a human-associated lineage, with many examples sourced from human extraintestinal infections. ST1193 from urban-adapted birds, wastewater, and companion animals are frequent, but isolates from animal agriculture are notably absent. Phylogenomic analysis identified several clades segregated by serogroup, all noted to carry highly similar F plasmids and antimicrobial resistance (AMR) signatures. Investigation of these plasmids revealed virulence regions with similarity to pUTI89, a key F virulence plasmid among dominant pandemic extraintestinal pathogenic E. coli lineages, and encoding a complex antibiotic resistance structure mobilized by IS26. This work has uncovered a series of F virulence plasmids in ST1193 and shows that the lineage mimics the host range and virulence attributes of other E. coli strains that carry pUTI89. These observations have significant ramifications for epidemiological source tracking of emerging and established pandemic ExPEC lineages.

Genomic characterisation of an entomopathogenic strain of Serratia ureilytica in the critically endangered phasmid Dryococelus australis.

Between 2014 and 2019, unexpected mortalities were observed in a colony of Dryococelus australis, an endangered stick-insect kept at the Melbourne Zoo for a breeding and conservation program. Pure cultures of Serratia spp. were obtained from the haemolymph of moribund and recently deceased individuals. The combined bacteriological and histopathological observations suggested an infectious cause of these mortalities. Genotyping of Serratia sp. isolated from the insects and their environment revealed a predominant strain profile. A representative isolate, AM923, was entirely sequenced and compared to 616 publicly available Serratia spp. genomes, including 37 associated with insects. The genomes were distributed into 3 distinct groups, with 63% of the insect-associated isolates within a single clade (clade A) containing AM923, separated from most environmental/plant-associated strains (clade B) and human isolates (clade C). Average nucleotide identity and phylogenetic analyses identified AM923 as S. ureilytica and revealed similarities with putatively entomopathogenic strains. An experimental infection model in honey bees (Apis mellifera) confirmed the pathogenic potential of AM923. A urease operon was found in most insect isolates and a PCR assay, based on the ureB gene sequence, was used to confirm the presence of AM923 in experimentally infected bees. This species-specific PCR could be applied to detect entomopathogenic Serratia spp. in infected insects or their environment.

Healthcare-associated infections caused by chlorhexidine-tolerant Serratia marcescens carrying a promiscuous IncHI2 multi-drug resistance plasmid in a veterinary hospital.

The bacterium Serratia marcescens can cause opportunistic infections in humans and in animals. In veterinary settings, the diversity, reservoirs and modes of transmission of this pathogen are poorly understood. The phenotypes and genotypes of Serratia spp. isolated from dogs, cats, horses, a bird and a rabbit examined at an Australian veterinary hospital between 2008 and 2019 were characterised. The isolates were identified as S. marcescens (n = 15) or S. ureilytica (n = 3) and were placed into four distinct phylogenetic groups. Nine quasi-clonal isolates associated with post-surgical complications in different patients displayed high levels of resistance to the antimicrobials fluoroquinolones, cephalosporins, aminoglycosides, and to the disinfectant chlorhexidine. A Serratia sp. with a similar resistance profile was also isolated from chlorhexidine solutions used across the Hospital, suggesting that these infections had a nosocomial origin. A genomic island encoding a homolog of the Pseudomonas MexCD-OprJ biocide efflux system was detected in the chlorhexidine-tolerant Serratia. The nine multi-drug resistant Serratia isolates also possessed a Ser-83-Ile mutation in GyrA conferring fluoroquinolone resistance, and carried a large IncHI2 conjugative plasmid encoding antimicrobial and heavy metal resistances. This replicon was highly similar to a plasmid previously detected in a strain of Enterobacter hormaechei recovered from the Hospital environment. IncHI2 plasmids are commonly found in Enterobacteriaceae, but are rarely present in Serratia spp., suggesting that this plasmid was acquired from another organism. A chlorhexidine-tolerant Serratia isolate which lacked the IncHI2 plasmid was used in mating experiments to demonstrate the transfer of multi-drug resistance from a E. hormaechei donor. This study illustrates the importance of environmental surveillance of biocide-resistance in veterinary hospitals.

Genomic comparisons of Escherichia coli ST131 from Australia.

Escherichia coli ST131 is a globally dispersed extraintestinal pathogenic E. coli lineage contributing significantly to hospital and community acquired urinary tract and bloodstream infections. Here we describe a detailed phylogenetic analysis of the whole genome sequences of 284 Australian ST131 E. coli isolates from diverse sources, including clinical, food and companion animals, wildlife and the environment. Our phylogeny and the results of single nucleotide polymorphism (SNP) analysis show the typical ST131 clade distribution with clades A, B and C clearly displayed, but no niche associations were observed. Indeed, interspecies relatedness was a feature of this study. Thirty-five isolates (29 of human and six of wild bird origin) from clade A (32 fimH41, 2 fimH89, 1 fimH141) were observed to differ by an average of 76 SNPs. Forty-five isolates from clade C1 from four sources formed a cluster with an average of 46 SNPs. Within this cluster, human sourced isolates differed by approximately 37 SNPs from isolates sourced from canines, approximately 50 SNPs from isolates from wild birds, and approximately 52 SNPs from isolates from wastewater. Many ST131 carried resistance genes to multiple antibiotic classes and while 41 (14 %) contained the complete class one integron-integrase intI1, 128 (45 %) isolates harboured a truncated intI1 (462-1014 bp), highlighting the ongoing evolution of this element. The module intI1-dfrA17-aadA5-qacEΔ1-sul1-ORF-chrA-padR-IS1600-mphR-mrx-mphA, conferring resistance to trimethoprim, aminoglycosides, quaternary ammonium compounds, sulphonamides, chromate and macrolides, was the most common structure. Most (73 %) Australian ST131 isolates carry at least one extended spectrum ß-lactamase gene, typically blaCTX-M-15 and blaCTX-M-27. Notably, dual parC-1aAB and gyrA-1AB fluoroquinolone resistant mutations, a unique feature of clade C ST131 isolates, were identified in some clade A isolates. The results of this study indicate that the the ST131 population in Australia carries diverse antimicrobial resistance genes and plasmid replicons and indicate cross-species movement of ST131 strains across diverse reservoirs.

Colonization of a hand washing sink in a veterinary hospital by an Enterobacter hormaechei strain carrying multiple resistances to high importance antimicrobials.

BACKGROUND: Hospital intensive care units (ICUs) are known reservoirs of multidrug resistant nosocomial bacteria. Targeted environmental monitoring of these organisms in health care facilities can strengthen infection control procedures. A routine surveillance of extended spectrum beta-lactamase (ESBL) producers in a large Australian veterinary teaching hospital detected the opportunistic pathogen Enterobacter hormaechei in a hand washing sink of the ICU. The organism persisted for several weeks, despite two disinfection attempts. Four isolates were characterized in this study. METHODS: Brilliance-ESBL selective plates were inoculated from environmental swabs collected throughout the hospital. Presumptive identification was done by conventional biochemistry. Genomes of multidrug resistant Enterobacter were entirely sequenced with Illumina and Nanopore platforms. Phylogenetic markers, mobile genetic elements and antimicrobial resistance genes were identified in silico. Antibiograms of isolates and transconjugants were established with Sensititre microdilution plates. RESULTS: The isolates possessed a chromosomal Tn7-associated silver/copper resistance locus and a large IncH12 conjugative plasmid encoding resistance against tellurium, arsenic, mercury and nine classes of antimicrobials. Clusters of antimicrobial resistance genes were associated with class 1 integrons and IS26, IS903 and ISCR transposable elements. The blaSHV-12, qnrB2 and mcr-9.1 genes, respectively conferring resistance to cephalosporins, quinolones and colistin, were present in a locus flanked by two IS903 copies. ESBL production and enrofloxacin resistance were confirmed phenotypically. The isolates appeared susceptible to colistin, possibly reflecting the inducible nature of mcr-9.1. CONCLUSIONS: The persistence of this strain in the veterinary hospital represented a risk of further accumulation and dissemination of antimicrobial resistance, prompting a thorough disinfection of the ICU. The organism was not recovered from subsequent environmental swabs, and nosocomial Enterobacter infections were not observed in the hospital during that period. This study shows that targeted routine environmental surveillance programs to track organisms with major resistance phenotypes, coupled with disinfection procedures and follow-up microbiological cultures are useful to control these risks in sensitive areas of large veterinary hospitals.

Exploration of antibiotic resistance risks in a veterinary teaching hospital with Oxford Nanopore long read sequencing.

The Oxford Nanopore MinION DNA sequencing device can produce large amounts of long sequences, typically several kilobases, within a few hours. This long read capacity was exploited to detect antimicrobial resistance genes (ARGs) in a large veterinary teaching hospital environment, and to assess their taxonomic origin, genetic organisation and association with mobilisation markers concurrently. Samples were collected on eight occasions between November 2016 and May 2017 (inclusive) in a longitudinal study. Nanopore sequencing was performed on total DNA extracted from the samples after a minimal enrichment step in broth. Many ARGs present in the veterinary hospital environment could potentially confer resistance to antimicrobials widely used in treating infections of companion animals, including aminoglycosides, extended-spectrum beta-lactams, sulphonamides, macrolides, and tetracyclines. High-risk ARGs, defined here as single or multiple ARGs associated with pathogenic bacterial species or with mobile genetic elements, were shared between the intensive care unit (ICU) patient cages, a dedicated laundry trolley and a floor cleaning mop-bucket. By contrast, a floor surface from an office corridor without animal contact and located outside the veterinary hospital did not contain such high-risk ARGs. Relative abundances of high-risk ARGs and co-localisation of these genes on the same sequence read were higher in the laundry trolley and mop bucket samples, compared to the ICU cages, suggesting that amplification of ARGs is likely to occur in the collection points for hospital waste. These findings have prompted the implementation of targeted intervention measures in the veterinary hospital to mitigate the risks of transferring clinically important ARGs between sites and to improve biosecurity practices in the facility.

Whole genome sequence analysis of Australian avian pathogenic Escherichia coli that carry the class 1 integrase gene.

Avian pathogenic Escherichia coli (APEC) cause widespread economic losses in poultry production and are potential zoonotic pathogens. Genome sequences of 95 APEC from commercial poultry operations in four Australian states that carried the class 1 integrase gene intI1, a proxy for multiple drug resistance (MDR), were characterized. Sequence types ST117 (22/95), ST350 (10/95), ST429 and ST57 (each 9/95), ST95 (8/95) and ST973 (7/95) dominated, while 24 STs were represented by one or two strains. FII and FIB repA genes were the predominant (each 93/95, 98 %) plasmid incompatibility groups identified, but those of B/O/K/Z (25/95, 26 %) and I1 (24/95, 25 %) were also identified frequently. Virulence-associated genes (VAGs) carried by ColV and ColBM virulence plasmids, including those encoding protectins [iss (91/95, 96 %), ompT (91/95, 96 %) and traT (90/95, 95 %)], iron-acquisition systems [sitA (88/95, 93 %), etsA (87/95, 92 %), iroN (84/95, 89 %) and iucD/iutA (84/95, 89 %)] and the putative avian haemolysin hylF (91/95, 96 %), featured prominently. Notably, mobile resistance genes conferring resistance to fluoroquinolones, colistin, extended-spectrum ß-lactams and carbapenems were not detected in the genomes of these 95 APEC but carriage of the sulphonamide resistance gene, sul1 (59/95, 63 %), the trimethoprim resistance gene cassettes dfrA5 (48/95, 50 %) and dfrA1 (25/95, 27 %), the tetracycline resistance determinant tet(A) (51/95, 55 %) and the ampicillin resistance genes blaTEM-1A/B/C (48/95, 52 %) was common. IS26 (77/95, 81 %), an insertion element known to capture and mobilize a wide spectrum of antimicrobial resistance genes, was also frequently identified. These studies provide a baseline snapshot of drug-resistant APEC in Australia and their role in the carriage of ColV-like virulence plasmids.

A Novel Glaesserella sp. Isolated from Pigs with Severe Respiratory Infections Has a Mosaic Genome with Virulence Factors Putatively Acquired by Horizontal Transfer.

An unknown member of the family Pasteurellaceae was repeatedly isolated from 20- to 24-week-old pigs with severe pulmonary lesions reared on the same farm in Victoria, Australia. The etiological diagnosis of the disease was inconclusive. The complete genome sequence analysis of one strain, 15-184, revealed some phylogenic proximity to Glaesserella (Haemophilus) parasuis, the cause of Glasser's disease. However, the sequences of the 16S rRNA and housekeeping genes, as well as the average nucleotide identity scores, differed from those of all other known species in the family Pasteurellaceae The protein content of 15-184 was composite, with 60% of coding sequences matching known G. parasuis products, while more than 20% had a closer relative in the genera Actinobacillus, Mannheimia, Pasteurella, and Bibersteinia Several putative virulence genes absent from G. parasuis but present in other Pasteurellaceae were also found, including the apxIII RTX toxin gene from Actinobacillus pleuropneumoniae, ABC transporters from Actinobacillus minor, and iron transporters from various species. Three prophages and one integrative conjugative element were present in the isolate. Horizontal gene transfers might explain the mosaic genomic structure and atypical metabolic and virulence characteristics of 15-184. This organism has not been assigned a taxonomic position in the family, but this study underlines the need for a large-scale epidemiological and clinical characterization of this novel pathogen in swine populations, as a genomic analysis suggests it could have a severe impact on pig health.IMPORTANCE Several species of Pasteurellaceae cause a range of significant diseases in pigs. A novel member of this family was recently isolated from Australian pigs suffering from severe respiratory infections. Comparative whole-genome analyses suggest that this bacterium represents a new species, which possesses a number of virulence genes horizontally acquired from a diverse range of other Pasteurellaceae While the possible contribution of other coinfecting noncultivable agents to the disease has not been ruled out in this study, the repertoire of virulence genes found in this organism may nevertheless explain some aspects of the associated pathology observed on the farm. The prevalence of this novel pathogen within pig populations is currently unknown. This finding is of particular importance for the pig industry, as this organism can have a serious impact on the health of these animals.

Pathological and microbiological investigations into cases of bacterial chondronecrosis and osteomyelitis in broiler poultry.

Bacterial chondronecrosis and osteomyelitis (BCO) is increasingly recognized as a major cause of lameness in commercial broilers chickens worldwide, but the pathogenesis of the condition is incompletely understood. This was a longitudinal study of 20 commercial broiler farms in Victoria, Australia, to investigate the aetiology and pathology of BCO. Thorough postmortem examination was performed on culled and dead birds (n = 325) from 20 different flocks at either 1 week, 4 weeks or 5 weeks of age and samples were analysed by conventional bacteriology, molecular identification of infectious organisms detected, serology and histopathology. BCO occurs throughout the life of broiler flocks at a very high rate, with lesions detected in 28% (95% CI 23-34%) of the mortalities and culls. The condition occurs with similar prevalence in both the femur and tibiotarsus. BCO is an infectious process that appears to result from bacteraemia and haematological spread of bacterial pathogens, especially Escherichia coli, to the bones, with 65.3% bacterial isolates from histologically confirmed BCO identified as E. coli, 11.5% as Staphylococcus and the remainder composed of mixed infections or a range of other minor isolates. We observed that almost all E. coli isolated from cases of BCO are avian pathogenic E. coli, suggesting that preventative measures should be directed at this organism.

Fructan extracts from wheat stem and barley grain stimulate large bowel fermentation in rats.

The benefits of inulin-type fructans for bowel health are well established, but less so for other fructan sources. In vitro data suggest that fructans extracted from cereals are readily fermented and produce favorable short-chain fatty acid profiles; however, whether this occurs in vivo is unknown. We hypothesized that in rats, fructans extracted from wheat stem and barley grain would have similar effects on fermentation as oligofructose (OF). Fifty-six male Sprague-Dawley rats were randomly assigned to 1 of 7 dietary treatments that contained either 2% or 5% fructan, provided by a barley grain fructan extract (BGFE), a wheat stem fructan extract, or OF or no added fructan (control). The duration of the feeding study was 14 days. Rats fed diets containing 5% fructan had higher cecal digesta weights; larger acetate, propionate, and total short-chain fatty acid pools; and lower pHs in comparison with the control group. In addition, only the 5% OF and 5% BGFE groups increased cecal butyrate pools, and 5% BGFE was the only group in which colonic digesta pH was lower than that of the control. Diets containing 2% fructan did not affect any of these fermentation end points. Whereas bifidobacteria numbers in cecal digesta of 2% and 5% OF were higher than that in the control group, they were not different from those in rats fed diets containing BGFE and wheat stem fructan extract. Barley grain and wheat stem fructans produced similar large bowel fermentation patterns to OF when fed to rats at 5% of the diet.