Applied and Environmental Microbiology
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Published By American Society For Microbiology

1098-5336, 0099-2240
Updated Saturday, 16 October 2021

2021 ◽  
Vol 87 (21) ◽  

Shuaishuai Xie ◽  
Yahong Tan ◽  
Wenxia Song ◽  
Weican Zhang ◽  
Qingsheng Qi ◽  

Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the phylum Bacteroidetes . It digests crystalline cellulose with an unknown mechanism, and possesses a type IX secretion system (T9SS) that can recognize the C-terminal domain (CTD) of the cargo protein as a signal. In this study, the functions of CTD in the secretion and localization of T9SS substrates in C. hutchinsonii were studied by fusing the green fluorescent protein (GFP) with CTD from CHU_2708. CTD is necessary for the secretion of GFP by C. hutchinsonii T9SS. The GFP-CTD CHU_2708 fusion protein was found to be glycosylated in the periplasm with a molecular mass about 5 kDa higher than that predicted from its sequence. The glycosylated protein was sensitive to peptide- N -glycosidase F which can hydrolyze N -linked oligosaccharides. Analyses of mutants obtained by site-directed mutagenesis of asparagine residues in the N-X-S/T motif of CTD CHU_2708 suggest that N -glycosylation occurred on the CTD. CTD N- glycosylation is important for the secretion and localization of GFP-CTD recombinant proteins in C. hutchinsonii . Glycosyltransferase encoding gene chu_3842 , a homologous gene of Campylobacter jejuni pglA , was found to participate in the N -glycosylation of C. hutchinsonii . Deletion of chu_3842 affected cell motility, cellulose degradation, and cell resistance to some chemicals. Our study provided the evidence that CTD as the signal of T9SS was N -glycosylated in the periplasm of C. hutchinsonii . IMPORTANCE The bacterial N -glycosylation system has previously only been found in several species of Proteobacteria and Campylobacterota , and the role of N -linked glycans in bacteria is still not fully understood. C. hutchinsonii has a unique cell-contact cellulose degradation mechanism, and many cell surface proteins including cellulases are secreted by the T9SS. Here, we found that C. hutchinsonii , a member of the phylum Bacteroidetes , has an N -glycosylation system. Glycosyltransferase CHU_3842 was found to participate in the N -glycosylation of C. hutchinsonii proteins, and had effects on cell resistance to some chemicals, cell motility, and cellulose degradation. Moreover, N -glycosylation occurs on the CTD translocation signal of T9SS. The glycosylation of CTD apears to play an important role in affecting T9SS substrates transportation and localization. This study enriched our understanding of the widespread existence and multiple biological roles of N -glycosylation in bacteria.

Siobhán C. McCarthy ◽  
Guerrino Macori ◽  
Gina Duggan ◽  
Catherine M. Burgess ◽  
Séamus Fanning ◽  

Shiga toxin-producing Escherichia coli (STEC) are a diverse group of pathogenic bacteria capable of causing serious human illness and serogroups O157 and O26 are frequently implicated in human disease. Ruminant hosts are the primary STEC reservoir and small ruminants are important contributors to STEC transmission. This study investigated the prevalence, serotypes and shedding dynamics of STEC, including the super-shedding of serogroups O157 and O26, in Irish sheep. Recto-anal mucosal swab samples (N=840) were collected over 24 months from two ovine slaughtering facilities. Samples were plated on selective agars and were quantitatively and qualitatively assessed via real-time PCR for Shiga-toxin prevalence and serogroup. A subset of STEC isolates (N=199) were selected for whole-genome sequencing and analysed in silico . In total, 704/840 (83.8%) swab samples were Shiga-toxin positive following RT-PCR screening, and 363/704 (51.6%) animals were subsequently culture positive for STEC. Five animals were shedding STEC O157 and three of these were identified as super-shedders. No STEC O26 was isolated. Post-hoc statistical analysis showed that younger animals are more likely to harbour STEC and STEC carriage is most prevalent during the summer months. Following sequencing, 178/199 genomes were confirmed as STEC. Thirty-five different serotypes were identified, fifteen of which were not yet reported in sheep. Serotype O91:H14 was the most frequently reported. Eight Shiga-toxin gene variants were reported, two stx 1 and six stx 2 , and three novel Shiga-toxin subunit combinations were observed. Variant stx 1c was the most prevalent, while many strains also harboured stx 2b . Importance Shiga toxin-producing Escherichia coli (STEC) are foodborne, zoonotic pathogens of significant public health concern. All STEC harbour stx , a critical virulence determinant, but it is not expressed in most serotypes. Sheep shed the pathogen via faecal excretion and are increasingly recognised as important contributors in the dissemination of STEC. In this study, we have found that there is high prevalence of STEC circulating within sheep and prevalence is related to animal age and seasonality. Further, sheep harbour a variety of non-O157 STEC, whose prevalence and contribution to human disease has been under investigated for many years. A variety of Stx variants were also observed, some of which are of high clinical importance.

Carina M. Hall ◽  
Anthony L. Baker ◽  
Jason W. Sahl ◽  
Mark Mayo ◽  
Holger C. Scholz ◽  

Distinct Burkholderia strains were isolated from soil samples collected in tropical northern Australia (Northern Territory and the Torres Strait Islands, Queensland). Phylogenetic analysis of 16S rRNA and whole genome sequences revealed these strains were distinct from previously described Burkholderia species and assigned them to two novel clades within the B. pseudomallei complex (Bpc). Because average nucleotide identity and digital DNA-DNA hybridization calculations are consistent with these clades representing distinct species, we propose the names Burkholderia mayonis sp. nov. and Burkholderia savannae sp. nov. Strains assigned to B. mayonis sp. nov. include type strain BDU6 T (=TSD-80; LMG 29941; ASM152374v2) and BDU8. Strains assigned to B. savannae sp. nov. include type strain MSMB266 T (=TSD-82; LMG 29940; ASM152444v2), MSMB852, BDU18, and BDU19. Comparative genomics revealed unique coding regions for both putative species, including clusters of orthologous genes associated with phage. Type strains of both B. mayonis sp. nov. and B. savannae sp. nov. yielded biochemical profiles distinct from each other and other species in the Bpc, and profiles also varied among strains within B. mayonis sp. nov. and B. savannae sp. nov. Matrix-assisted laser desorption ionization–time of flight analysis revealed a B. savannae sp. nov. cluster separate from other species, whereas B. mayonis sp. nov. strains did not form a distinct cluster. Neither B. mayonis sp. nov. nor B. savannae sp. nov. caused mortality in mice when delivered via the subcutaneous route. The addition of B. mayonis sp. nov. and B. savannae sp. nov. results in eight species currently in the Bpc. IMPORTANCE Burkholderia species can be important sources of novel natural products and new species are of interest to diverse scientific disciplines. Although many Burkholderia species are saprophytic, Burkholderia pseudomallei is the causative agent of the disease melioidosis. Understanding the genomics and virulence of the closest relatives to B. pseudomallei ( i.e., the other species within the Bpc) is important for identifying robust diagnostic targets specific to B. pseudomallei and understanding evolution of virulence in B. pseudomallei . Two proposed novel species, B. mayonis sp. nov. and B. savannae sp. nov., were isolated from soil samples collected from multiple locations in northern Australia. The two proposed species belong to the Bpc but are phylogenetically distinct from all other members of this complex. The addition of B. mayonis sp. nov. and B. savannae sp. nov. results in a total of eight species within this significant complex of bacteria that are available for future studies.

Carissa A. Odland ◽  
Roy Edler ◽  
Noelle R. Noyes ◽  
Scott A. Dee ◽  
Joel Nerem ◽  

A longitudinal study was conducted to assess the impact of different antimicrobial exposures of nursery-phase pigs on patterns of phenotypic antimicrobial resistance in fecal indicator organisms throughout the growing phase. Based on practical approaches used to treat moderate to severe PRRSV-associated secondary bacterial infections, two antimicrobial protocols of differing intensity of exposure [44.1 and 181.5 animal-treatment days per 1000 animal days at risk (ATD)] were compared with a control group with minimal antimicrobial exposure (2.1 ATD). Litter-matched pigs (n = 108) with no prior antimicrobial exposure were assigned randomly to the treatment groups. Pen fecal samples were collected nine times during the wean-to-finish period and cultured for Escherichia coli and Enterococcus spp. Antimicrobial susceptibility testing was conducted using NARMS gram-negative and gram-positive antibiotic panels. Despite up to 65-fold difference in ATD, few and modest differences were observed between groups and over time. Resistant patterns at marketing overall remained similar to those observed at weaning, prior to any antimicrobial exposures. Those differences observed could not readily be reconciled with the patterns of antimicrobial exposure. Resistance of E. coli to streptomycin was higher in the group exposed to 44.1 ATD, but no aminoglycosides were used. In all instances where resistance differed between time points, the higher resistance occurred early in the trial prior to any antimicrobial exposures. These minimal impacts on AMR despite substantially different antimicrobial exposures point to the lack of understanding of the drivers of AMR at the population level and the likely importance of factors other than antimicrobial exposure. IMPORTANCE Despite a recognized need for more longitudinal studies to assess the effects of antimicrobial use on resistance in food animals, they remain sparse in the literature, and most longitudinal studies of pigs have been observational. The current experimental study had the advantages of greater control of potential confounding, precise measurement of antimicrobial exposures which varied markedly between groups and tracking of pigs until market age. Overall, resistance patterns were remarkably stable between the treatment groups over time, and the differences observed could not be readily reconciled with the antimicrobial exposures, indicating the likely importance of other determinants of AMR at the population level.

Yu Cao ◽  
Katherine Dever ◽  
Sathesh Kumar Sivasankaran ◽  
Scott V. Nguyen ◽  
Guerrino Macori ◽  

Cronobacter sakazakii is a typical example of a xerotolerant bacterium. It is epidemiologically linked to low moisture foods like powdered infant formula (PIF) and is associated with high fatality rates among neonates. We characterized the xerotolerance in a clinically isolated strain, C. sakazakii ATCC™29544 T , and compared the desiccation tolerance with an environmental strain, C. sakazakii SP291, whose desiccation tolerance was previously characterized. We found that, although the clinical strain was desiccation-tolerant, the level of tolerance was compromised when compared to the environmental strain. RNA-seq based deep transcriptomic characterization identified a unique transcriptional profile in the clinical strain compared to what was already known for the environmental strain. As RNA-seq was also carried out in different TSB growth conditions, genes that were expressed specifically under desiccated conditions were identified and denoted as desiccation responsive genes (DRGs). Interestingly, these DRGs included transcriptomic factors like fnr , ramA, and genes associated with inositol metabolism, a phenotype as yet unreported in C. sakazakii . Further, the clinical strain did not express the proP gene, which was previously reported to be very important for desiccation survival and persistence. Interestingly, analysis of the plasmid genes showed that the iron metabolism in desiccated C. sakazakii ATCC™29544 T cells specifically involved the siderophore cronobactin encoded by the iucABCD genes. Confirmatory studies using qRT-PCR determined that, though the secondary desiccation response genes were upregulated in C. sakazakii ATCC™29544 T , the level of up-regulation was lower compared to that in C. sakazakii SP291. All these factors could collectively contribute to the compromised desiccation tolerance in the clinical strain. IMPORTANCE Cronobacter sakazakii has in past led to outbreaks, particularly associated with food that are low in moisture content. This species has adapted to survive in low water conditions and can survive in such environments for long periods. These characteristics have enabled the pathogen to contaminate powder infant formula, a food matrix with which the pathogen has been epidemiologically associated. Even though clinically adapted strains can also be isolated, there is no information on how the clinical strains adapt to low moisture environments. Our research assessed the adaptation of a clinically isolated strain to low moisture survival on sterile stainless steel coupons and compared the survival to a highly desiccation-tolerant environmental strain. We found that, even though the clinical strain is desiccation-tolerant, the rate of tolerance was compromised compared to the environmental strain. A deeper investigation using RNA-seq identified that the clinical strain used pathways different from that of the environmental strain to adapt to low moisture conditions. This shows that the adaptation to desiccation conditions, at least for C. sakazakii , is strain-specific and that different strains have used different evolutionary strategies for adaptation.

Rui Xu ◽  
Duanyi Huang ◽  
Xiaoxu Sun ◽  
Miaomiao Zhang ◽  
Dongbo Wang ◽  

Biological arsenite (As(III)) oxidation is an important process in the removal of toxic arsenic (As) from contaminated water. However, the diversity and metabolic potentials of As(III)-oxidizing bacteria (AOBs) responsible for As(III) oxidation in wastewater treatment facilities are not well documented. In this study, two groups of bioreactors inoculated with activated sludge were operated under anoxic or oxic conditions to treat As-containing synthetic wastewater. Batch tests of inoculated sludges from the bioreactors further indicated that microorganisms could use nitrate or oxygen as electron acceptors to stimulate biological As(III) oxidation, suggesting the potentials of this process in wastewater treatment facilities. In addition, DNA-based stable isotope probing (DNA-SIP) was performed to identify the putative AOBs in the activated sludge. Bacteria associated with Thiobacillus were identified as nitrate-dependent AOBs, while bacteria associated with Hydrogenophaga were identified as aerobic AOBs in activated sludge. Metagenomic binning reconstructed a number of high-quality metagenome-assembled genomes (MAGs) associated with the putative AOBs. Functional genes encoding for As resistance, As(III) oxidation, denitrification, and carbon fixation were identified in these MAGs, suggesting their potentials for chemoautotrophic As(III) oxidation. In addition, the presence of genes encoding secondary metabolite biosynthesis and extracellular polymeric substance metabolism in these MAGs may facilitate the proliferation of these AOBs in activated sludge and enhance their capacity for As(III) oxidation. Importance AOBs play an important role in the removal of toxic arsenic from wastewater. Most of the AOBs have been isolated from natural environments. However, knowledge regarding the structure and functional roles of As(III)-oxidizing communities in wastewater treatment facilities are not well documented. The combination of DNA-SIP and metagenomic binning provides an opportunity to elucidate the diversity of in situ AOBs community inhabited the activated sludges. In this study, the putative AOBs responsible for As(III) oxidation in wastewater treatment facilities were identified, and their metabolic potentials including As(III) oxidation, denitrification, carbon fixation, secondary metabolites biosynthesis, and extracellular polymeric substances metabolisms were investigated. This observation provides an understanding of anoxic and/or oxic AOBs during the A(III) oxidation process in wastewater treatment facilities, which may contribute to the removal of As from contaminated water.

Hillary Adawo Onjong ◽  
Victor Ntuli ◽  
Joseph Wambui ◽  
Mercy Mwaniki ◽  
Patrick Murigu Kamau Njage

The current study was designed to evaluate the potential impact of the level of regulation on the prevalence and patterns of antimicrobial agent resistance in bacteria isolated from fish. The study sites included two large lakes and both semi-regulated and unregulated fish value chains. A total of 328 bacterial isolates belonging to 11 genera were evaluated for antimicrobial susceptibility testing using disk diffusion method. The bacteria species were tested against 12 different antibiotics (trimethoprim-sulfamethoxazole, tetracycline, ampicillin, cefotaxime, chloramphenicol, nalidixic acid, amoxicillin, meropenem, ciprofloxacin, nitrofurantoin, cefuroxime and kanamycin). Data analysis was done to assess the heterogeneity in proportion of resistant bacterial species within and between the two value chains using a random-effects model proposed by DerSimonian and Laird. Statistical heterogeneity within and between groups were estimated using the Cochran Chi -square test and the Cochrane- I 2 . Overall proportion of bacterial species resistant to antimicrobial agents in semi-regulated and unregulated value chains ranged from 0.00 to 0.88 and 0.09 to 0.95, respectively. Shigella spp. had the highest proportion of bacteria that were resistant against most of the antimicrobial agents used. The bacterial species were highly resistant to ampicillin and amoxicillin and the highest multi-drug resistance capacity was observed in Shigella spp. (18.3%, n = 328), Vibrio spp. (18.3%) and L. monocytogenes (12.2%). We observed strong heterogeneity within and between the two value chains regarding proportion of resistant bacterial species. Sun dried fish in both value chains had significantly high proportion of resistant bacterial species. Comparing the two value chains, unregulated value chain had significantly higher proportion of bacterial species that were resistant. In order to mitigate the risk of transmitting antimicrobial resistant bacteria to consumers along the fish value chain, good manufacturing practices coupled with identification and management of possible sources of contamination are recommended for fish and potentially other foods distributed along the less regulated value chains. Importance In order to mitigate the risk of transmitting antimicrobial resistant bacteria to consumers along the fish value chain, good manufacturing practices coupled with identification and management of possible sources of contamination are recommended for fish and potentially other foods distributed along the less regulated value chains.

Jason A. Rothman ◽  
Theresa B. Loveless ◽  
Joseph Kapcia ◽  
Eric D. Adams ◽  
Joshua A. Steele ◽  

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early-warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the COVID-19 pandemic to track disease burden through analyses of SARS-CoV-2 RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the healthcare system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven treatment plants (WTPs), collected August 2020 – January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically-important SARS-CoV-2 variants, along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. Importance: Wastewater based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the COVID-19 pandemic to track the spread of SARS-CoV-2 without adding burden to healthcare systems. In this study, we used metatranscriptomics and RT-ddPCR to assay RNA viruses across Southern California wastewater from August 2020 – January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single nucleotide variants through sequencing. Likewise, WTPs harbored different viromes, and we detected other human pathogens such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

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