This study indicates that colonization of the intestinal mucosa by highly invasive strains of F. nucleatum may be a useful biomarker for gastrointestinal disease.

We report the presence and diversity of Bartonella spp. in bats of 13 insectivorous and frugivorous species collected from various locations across Kenya. Bartonella isolates were obtained from 23 Eidolon helvum, 22 Rousettus aegyptiacus, 4 Coleura afra, 7 Triaenops persicus, 1 Hipposideros commersoni, and 49 Miniopterus spp. bats. Sequence analysis of the citrate synthase gene from the obtained isolates showed a wide assortment of Bartonella strains. Phylogenetically, isolates clustered in specific host bat species. All isolates from R. aegyptiacus, C. afra, and T. persicus bats clustered in separate monophyletic groups. In contrast, E. helvum and Miniopterus spp. bats harbored strains that clustered in several groups. Further investigation is needed to determine whether these agents are responsible for human illnesses in the region.

A large-scale, whole-genome comparison of Canadian Neisseria gonorrhoeae isolates with high-level cephalosporin MICs was used to demonstrate a genomic epidemiology approach to investigate strain relatedness and dynamics. Although current typing methods have been very successful in tracing short-chain transmission of gonorrheal disease, investigating the temporal evolutionary relationships and geographical dissemination of highly clonal lineages requires enhanced resolution only available through whole-genome sequencing (WGS). Phylogenomic cluster analysis grouped 169 Canadian strains into 12 distinct clades. While some N. gonorrhoeae multiantigen sequence types (NG-MAST) agreed with specific phylogenomic clades or subclades, other sequence types (ST) and closely related groups of ST were widely distributed among clades. Decreased susceptibility to extended-spectrum cephalosporins (ESC-DS) emerged among a group of diverse strains in Canada during the 1990s with a variety of nonmosaic penA alleles, followed in 2000/2001 with the penA mosaic X allele and then in 2007 with ST1407 strains with the penA mosaic XXXIV allele. Five genetically distinct ESC-DS lineages were associated with penA mosaic X, XXXV, and XXXIV alleles and nonmosaic XII and XIII alleles. ESC-DS with coresistance to azithromycin was observed in 5 strains with 23S rRNA C2599T or A2143G mutations. As the costs associated with WGS decline and analysis tools are streamlined, WGS can provide a more thorough understanding of strain dynamics, facilitate epidemiological studies to better resolve social networks, and improve surveillance to optimize treatment for gonorrheal infections. N eisseria gonorrhoeae is a Gram-negative diplococcus bacterium that causes gonorrhea infections. Gonorrhea is the second most reported bacterial sexually transmitted infection (STI) in Canada, with reported cases increasing from 15.5 per 100,000 in 1997 to 36.2 per 100,000 in 2012 (1), and approximately 106 million cases are estimated annually worldwide (2). N. gonorrhoeae bacteria have developed resistance against sulfonamides, penicillins, tetracyclines, and fluoroquinolones (3, 4), and current treatment options now include third-generation extended-spectrum cephalosporins (ESC), namely, cefixime (CFM) and ceftriaxone (CRO) (5). MIC creep has seen the modal MIC values rise between 2001 and 2010 in Canada from 0.016 g/ml to 0.125 g/ml and 0.063 g/ml for CFM and CRO, respectively (6). These results coincide with recent clinical reports of treatment failures to primarily CFM monotherapy in Canada (7,8) and additional global reports of high-level CRO MICs in isolates from Japan and Europe (9-13). Furthermore, isolates with decreased susceptibility to cephalosporins and coresistance to azithromycin (AZM), a recommended cotherapy (5), have recently been identified in Canada (14).Decreased susceptibility to extended-spectrum cephalosporins (ESC-DS) and resistance to AZM have been attributed to several molecular mechanisms. The primary mechanism for ESC-DS is modification of the ...

Background Clostridium difficile are Gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15–35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking.Methodology/Principal FindingsGenotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production.Conclusions/SignificanceThis is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.

Background Clostridium difficile is an anaerobic, Gram-positive bacterium that has been implicated as the leading cause of antibiotic-associated diarrhea. Metronidazole is currently the first-line treatment for mild to moderate C. difficile infections. Our laboratory isolated a strain of C. difficile with a stable resistance phenotype to metronidazole. A shotgun proteomics approach was used to compare differences in the proteomes of metronidazole-resistant and -susceptible isolates.Methodology/Principal FindingsNAP1 C. difficile strains CD26A54_R (Met-resistant), CD26A54_S (reduced- susceptibility), and VLOO13 (Met-susceptible) were grown to mid-log phase, and spiked with metronidazole at concentrations 2 doubling dilutions below the MIC. Peptides from each sample were labeled with iTRAQ and subjected to 2D-LC-MS/MS analysis. In the absence of metronidazole, higher expression was observed of some proteins in C. difficile strains CD26A54_S and CD26A54_R that may be involved with reduced susceptibility or resistance to metronidazole, including DNA repair proteins, putative nitroreductases, and the ferric uptake regulator (Fur). After treatment with metronidazole, moderate increases were seen in the expression of stress-related proteins in all strains. A moderate increase was also observed in the expression of the DNA repair protein RecA in CD26A54_R.Conclusions/SignificanceThis study provided an in-depth proteomic analysis of a stable, metronidazole-resistant C. difficile isolate. The results suggested that a multi-factorial response may be associated with high level metronidazole-resistance in C. difficile, including the possible roles of altered iron metabolism and/or DNA repair.

Vibrio parahaemolyticus is a leading cause of seafood-borne gastroenteritis worldwide. Virulence is commonly associated with the production of two toxins, thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). Although the majority of clinical isolates produce TDH and/or TRH, clinical samples lacking toxin genes have been identified. In the present study, we investigated the effects of V. parahaemolyticus on transepithelial resistance (TER) and paracellular permeability in Caco-2 cultured epithelial cells. We found that V. parahaemolyticus profoundly disrupts epithelial barrier function in Caco-2 cells and that this disruption occurs independently of toxin production. Clinical isolates with different toxin genotypes all led to a significant decrease in TER, which was accompanied by an increased flux of fluorescent dextran across the Caco-2 monolayer, and profound disruption of actin and the tight junction-associated proteins zonula occludin protein 1 and occludin. Purified TDH, even at concentrations eightfold higher than those produced by the bacteria, had no effect on either TER or paracellular permeability. We used lactate dehydrogenase release as a measure of cytotoxicity and found that this parameter did not correlate with the ability to disrupt tight junctions. As the effect on barrier function occurs independently of toxin production, we used PCR to determine the toxin genotypes of V. parahaemolyticus isolates obtained from both clinical and environmental sources, and we found that 5.6% of the clinical isolates were toxin negative. These data strongly indicate that the effect on tight junctions is not due to TDH and suggest that there are other virulence factors.

Peptoniphilus spp. are Gram-positive anaerobic cocci (GPAC) that were formerly classified in the genus Peptostreptococcus. This study describes 15 cases of Peptoniphilus spp. bloodstream infection (BSI) diagnosed from 2007 to 2011 using 16S rDNA sequencing in patients with pneumonia, pre-term delivery, soft tissue infection or colon or bladder disease. Seven out of 15 (47%) of these cases had polymicrobial BSIs. One of the isolates was closely related to P. duerdenii (EU526290), while the other 14 isolates were most closely related to a Peptoniphilus sp. reference strain (ATCC 29743) and P. hareii (Y07839). Peptoniphilus is a rare but important cause of BSI.

A computer-assisted, kinetics-based enzyme-linked immunosorbent assay was adapted for the detection of coronavirus antibodies in feline serum. An alkaline antigen diluent (carbonate-bicarbonate buffer, pH 9.6) used in initial experiments produced diffuse, nonspecific color reactions in both viral and control antigen cuvettes which were correlated, paradoxically, with coronavirus antibody levels in test sera. These interfering reactions were minimized by use of lower-pH antigen diluents such as water and phosphate-buffered saline. Background kinetics-based enzyme-linked immunosorbent assay reactivity directed against a noncoronaviral component of antigen tissue culture fluids could then detected in numerous sera, particularly in samples with lower titers. Much of this reactivity was shown to be associated with bovine gamma globulins in cell culture fluid. It was not serum lot or species specific, since a variety of bovine serum lots as well as individual lots of serum from other mammalian and avian species reacted. Reactivity was markedly reduced when cells for antigen preparation were grown in gamma globulin-free bovine serum. Generation of corrected slope values from the kinetics-based enzyme-linked immunosorbent assay made it possible to correct for residual background reactivity in individual test sera and thus eliminate a potentially major source of false-positive reactions. Collectively, these studies indicated that the control of nonspecific reactivity in feline coronavirus serology is absolutely essential to obtain useful estimates of specific antibody responses.