A Comparative Study on Antimicrobial Resistance in Escherichia coli Isolated from Channel Catfish and Related Freshwater Fish Species.

Antimicrobial resistance (AMR) trends in 114 generic Escherichia coli isolated from channel catfish and related fish species were investigated in this study. Of these, 45 isolates were from commercial-sized channel catfish harvested from fishponds in Alabama, while 69 isolates were from Siluriformes products, accessed from the U.S. Department of Agriculture Food Safety and Inspection Service' (FSIS) National Antimicrobial Resistance Monitoring System (NARMS) program. Antibiotic susceptibility testing and whole genome sequencing were performed using the GenomeTrakr protocol. Upon analysis, the fishpond isolates showed resistance to ampicillin (44%), meropenem (7%) and azithromycin (4%). The FSIS NARMS isolates showed resistance to tetracycline (31.9%), chloramphenicol (20.3%), sulfisoxazole (17.4%), ampicillin (5.8%) and trimethoprim-sulfamethoxazole, nalidixic acid, amoxicillin-clavulanic acid, azithromycin and cefoxitin below 5% each. There was no correlation between genotypic and phenotypic resistance in the fishpond isolates, however, there was in NARMS isolates for folate pathway antagonists: Sulfisoxazole vs. sul1 and sul2 (p = 0.0042 and p < 0.0001, respectively) and trimethoprim-sulfamethoxazole vs. dfrA16 and sul1 (p = 0.0290 and p = 0.013, respectively). Furthermore, correlations were found for tetracyclines: Tetracycline vs. tet(A) and tet(B) (p < 0.0001 each), macrolides: Azithromycin vs. mph(E) and msr(E) (p = 0.0145 each), phenicols: Chloramphenicol vs. mdtM (p < 0.0001), quinolones: Nalidixic acid vs. gyrA_S83L=POINT (p = 0.0004), and ß-lactams: Ampicillin vs. blaTEM-1 (p < 0.0001). Overall, we recorded differences in antimicrobial susceptibility testing profiles, phenotypic-genotypic concordance, and resistance to critically important antimicrobials, which may be a public health concern.

Salmonella enterica Serovar Typhimurium Isolates from Wild Birds in the United States Represent Distinct Lineages Defined by Bird Type.

Salmonella enterica serovar Typhimurium is typically considered a host generalist; however, certain isolates are associated with specific hosts and show genetic features of host adaptation. Here, we sequenced 131 S. Typhimurium isolates from wild birds collected in 30 U.S. states during 1978-2019. We found that isolates from broad taxonomic host groups including passerine birds, water birds (Aequornithes), and larids (gulls and terns) represented three distinct lineages and certain S. Typhimurium CRISPR types presented in individual lineages. We also showed that lineages formed by wild bird isolates differed from most isolates originating from domestic animal sources, and that genomes from these lineages substantially improved source attribution of Typhimurium genomes to wild birds by a machine learning classifier. Furthermore, virulence gene signatures that differentiated S. Typhimurium from passerines, water birds, and larids were detected. Passerine isolates tended to lack S. Typhimurium-specific virulence plasmids. Isolates from the passerine, water bird, and larid lineages had close genetic relatedness with human clinical isolates, including those from a 2021 U.S. outbreak linked to passerine birds. These observations indicate that S. Typhimurium from wild birds in the United States are likely host-adapted, and the representative genomic data set examined in this study can improve source prediction and facilitate outbreak investigation. IMPORTANCE Within-host evolution of S. Typhimurium may lead to pathovars adapted to specific hosts. Here, we report the emergence of disparate avian S. Typhimurium lineages with distinct virulence gene signatures. The findings highlight the importance of wild birds as a reservoir for S. Typhimurium and contribute to our understanding of the genetic diversity of S. Typhimurium from wild birds. Our study indicates that S. Typhimurium may have undergone adaptive evolution within wild birds in the United States. The representative S. Typhimurium genomes from wild birds, together with the virulence gene signatures identified in these bird isolates, are valuable for S. Typhimurium source attribution and epidemiological surveillance.

Mcc1229, an Stx2a-Amplifying Microcin, Is Produced In Vivo and Requires CirA for Activity.

Enterohemorrhagic Escherichia coli (EHEC) strains, including the foodborne pathogen E. coli O157:H7, are responsible for thousands of hospitalizations each year. Various environmental triggers can modulate pathogenicity in EHEC by inducing the expression of Shiga toxin (Stx), which is encoded on a lambdoid prophage and transcribed together with phage late genes. Cell-free supernatants of the sequence type 73 (ST73) E. coli strain 0.1229 are potent inducers of Stx2a production in EHEC, suggesting that 0.1229 secretes a factor that activates the SOS response and leads to phage lysis. We previously demonstrated that this factor, designated microcin 1229 (Mcc1229), was proteinaceous and plasmid-encoded. To further characterize Mcc1229 and support its classification as a microcin, we investigated its regulation, determined its receptor, and identified loci providing immunity. The production of Mcc1229 was increased upon iron limitation, as determined by an enzyme-linked immunosorbent assay (ELISA), lacZ fusions, and quantitative real-time PCR (qRT-PCR). Spontaneous Mcc1229-resistant mutants and targeted gene deletion revealed that CirA was the Mcc1229 receptor. TonB, which interacts with CirA in the periplasm, was also essential for Mcc1229 import. Subcloning of the Mcc1229 plasmid indicated that Mcc activity was neutralized by two open reading frames (ORFs), each predicted to encode a domain of unknown function (DUF)-containing protein. In a germfree mouse model of infection, colonization with 0.1229 suppressed subsequent colonization by EHEC. Although Mcc1229 was produced in vivo, it was dispensable for colonization suppression. The regulation, import, and immunity determinants identified here are consistent with features of other Mccs, suggesting that Mcc1229 should be included in this class of small molecules.

Whole-Genome Sequences of Salmonella Isolates from an Ecological Wastewater Treatment System.

Twenty-seven Salmonella isolates were collected from four locations within an ecological wastewater treatment system located at The Pennsylvania State University and were subjected to whole-genome sequencing. The sequences obtained were used for in silico characterization, including serotyping and phylogenetic relatedness analysis.

A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression.

Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

Botulinum neurotoxin-encoding plasmids can be conjugatively transferred to diverse clostridial strains.

Most Group I Clostridium botulinum strains harbor botulinum neurotoxin (bont) genes on their chromosome, while some carry these genes (including bont/a, bont/b, and bont/f) on large plasmids. Prior work in our laboratory demonstrated that Group I BoNT plasmids were mobilized to C. botulinum recipient strains containing the Tn916 transposon. Here, we show that Tn916 is nonessential for plasmid transfer. Relying on an auxotrophic donor phenotype and a plasmid-borne selectable marker, we observed the transfer of pCLJ, a 270 kb plasmid harboring two bont genes, from its host strain to various clostridia. Transfer frequency was greatest to other Group I C. botulinum strains, but the plasmid was also transferred into traditionally nontoxigenic species, namely C. sporogenes and C. butyricum. Expression and toxicity of BoNT/A4 was confirmed in transconjugants by immunoblot and mouse bioassay. These data indicate that conjugation within the genus Clostridium can occur across physiological Groups of C. botulinum, supporting horizontal gene transfer via bont-bearing plasmids. The transfer of plasmids possessing bont genes to resistant Clostridium spp. such as C. sporogenes could impact biological safety for animals and humans. These plasmids may play an environmental role in initiating death in vertebrates, leading to decomposition and nutrient recycling of animal biomass.

Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain.

Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated "type H," has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 10(7) mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a "treatment-resistant" and highly potent toxin. However, the toxin's chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons.

Resveratrol is cidal to both classes of Haemophilus ducreyi.

Resveratrol, a polyphenolic phytoalexin, is produced by plants in response to infection and has antibacterial activity. Haemophilus ducreyi is a Gram-negative bacterium that is the causative agent of the sexually transmitted disease chancroid. This study employed minimum cidal concentration (MCC) assays to evaluate the potential of resveratrol as a microbicide against H. ducreyi. Five class I and four class II strains of H. ducreyi tested had MCCs ≤500 µg/mL. Resveratrol was also tested against Lactobacillus spp., part of the natural vaginal flora. Representative strains of Lactobacillus were co-cultured with H. ducreyi and 500 µg/mL resveratrol; in all cases, Lactobacillus was recovered in greater numbers than H. ducreyi. These results show that resveratrol is not only bacteriostatic but is bactericidal to H. ducreyi, confirming the compound's potential for use as a topical microbicide to prevent chancroid.