Grand Challenges in Terrestrial Microbiology

Understanding the functional role of different microbial populations is essential to ascertain whether environmental factors affecting their diversity, activity, and physiology will impact the functioning of terrestrial ecosystems. The soil environment represents one of the largest reservoirs of microbes in the biosphere and is the most significant in linking the activity of humans with the interaction and alteration of the major biogeochemical cycles. The global nitrogen cycle has been massively accelerated through the annual removal of over 100 Tg of atmospheric nitrogen for the production and use of fertilizers (Grube

The Characterisation of Antibiotic Resistance Plasmids in Escherichia coli and Klebsiella pneumoniae Isolates from Hospital and Community Settings

Thesis (MSc)--Stellenbosch University, 2021.ENGLISH ABSTRACT: Antimicrobial resistance has become one of the biggest challenges and threats to public health systems worldwide. Widespread distribution of resistance is commonly due to horizontal gene transfer, which includes mobile genetic elements (MGE) such as plasmids, insertion sequences, transposons, and integrons. This study aimed to characterise plasmids conferring antibiotic resistance in extended-spectrum β-lactamase (ESBL) positive Escherichia coli and Klebsiella pneumoniae isolates from bloodstream infections to determine whether ESBL and plasmid-mediated quinolone resistance (PMQR) genes were mobilised on the same plasmids and whether the same plasmids are disseminated in healthcare and community settings. Methods Illumina MiSeq whole-genome sequencing (WGS) was previously performed on 112 E. coli and 66 K. pneumoniae isolates from blood cultures submitted to the National Health Laboratory Service Microbiology Laboratory at Tygerberg Hospital during 2017 and 2018. Assembled genomes were interrogated for the presence of ESBL and PMQR genes and plasmid replicon types. Based on the results, eight E. coli and nine K. pneumoniae isolates were selected for plasmid sequencing on the Oxford Nanopore Technologies MinION platform. Unicycler assembler was used for hybrid assembly of Illumina short-reads and Nanopore plasmid long-reads. In silico analyses were performed using ResFinder, PlasmidFinder and ISsaga to identify ESBL and PMQR genes, plasmid replicon types, and MGEs. Results Based on Illumina WGS, the ESBL and PMQR containing isolates contained multiple resistance genes and IncF plasmid replicons, individually or in combination with additional plasmid types. The IncF replicons and resistance genes were on separate contigs, therefore associations between different IncF replicons and with resistance genes could not be confirmed. Nanopore sequencing resolved plasmids from several E. coli and K. pneumoniae isolates; however, chromosomal genes could not be visualised and misassembly resulted in fragmented plasmids. Hybrid assembly fully resolved plasmids and chromosomal genes in several E. coli and K. pneumoniae isolates. Amongst the E. coli isolates, three F-type multireplicon plasmids and two single replicon plasmids IncI1-γ and IncB/O/K/Z, which contained resistance genes, were described. Novel multireplicon plasmid FII(FIC)-FIB-X was detected and harboured ESBL blaTEM-135 and PMQR qnrS1. The blaCTX-M genes were confirmed to be chromosomally located in three E. coli isolates and plasmid-mediated on F-type plasmids in two E. coli isolates. K. pneumoniae isolates harboured single replicon F-type plasmids, multireplicon FIB-HI1B fusion plasmids, and a single replicon IncC plasmid. The FIB-HI1B plasmids were associated with blaCTX-M-15, aac(6’)-Ib-cr, and qnrS1. The blaCTX-M-15 was plasmid-mediated in all K. pneumoniae isolates. Conclusion Amongst the E. coli isolates, ESBL and PMQR genes were present both on the same plasmid and on separate plasmids. In K. pneumoniae, ESBLs and PMQRs were found collectively on the same plasmids, and the F-type plasmids harbouring ESBL and PMQR genes differed from those in E. coli. As only two community-acquired K. pneumoniae isolates were selected for Nanopore plasmid sequencing, conclusions regarding the dissemination of K. pneumoniae plasmids in healthcare and community settings could not be made. Plasmids of the same FAB-types were detected amongst E. coli isolates of various sequence types and from both hospital- and community-settings, which is indicative of spread between these settings.AFRIKAANS OPSOMMING: Antimikrobiese weerstandigheid is tans een van die grootste uitdagings en bedreigings vir gemeenskapsgesondheid wêreldwyd. Die wydverspreide distribusie van weerstandigheid is dikwels a.g.v. horisontale geenoordrag, wat gefasiliteer word deur mobiele genetiese elemente (MGE) soos plasmiede, invoegingsvolgordes, transposons en integrons. Hierdie studie het gepoog om plasmiede te karakteriseer wat antibiotika weerstandigheidsgene oordra in verlengde-spektrum β-laktamase-positiewe Escherichia coli en Klebsiella pneumoniae isolate, om sodoende te bepaal of verlengde-spektrum β-laktamase en plasmied-gemedieërde kinoloon weerstandigheidsgene op dieselfde plasmiede gemobiliseer is en of hierdie plasmiede in gesondheidsinstellings en gemeenskappe versprei het. Metodes Illumina MiSeq heel-genoom volgordebepaling is voorheen op 112 E. coli en 66 K. pneumoniae isolate vanaf bloedkulture gedoen, wat ingestuur is na die National Health Laboratory Service se Mikrobiologie Laboratorium by Tygerberg Hospitaal gedurende 2017 en 2018. Saamgestelde genome is ondersoek om die voorkoms van verlengde-spektrum β-laktamase en plasmied-gemedieërde kinoloon weerstandigheidsgene, en plasmied replikon tipes te bepaal. Op grond van hierdie resultate is agt E. coli en nege K. pneumoniae isolate gekies vir plasmied volgordebepaling op die Oxford Nanopore Technologies MinION platform. Die Unicycler samesteller is gebruik vir hibriedsamestelling van Illumina kort-lees en Nanopore plasmied lang-lees volgordes. In silico analise is met die ResFinder, PlasmidFinder en ISsaga gedoen om verlengde-spektrum β-laktamase en plasmied-gemedieërde kinoloon weerstandigheidsgene, plasmied replikon tipes en MGE te identifiseer. Resultate Op grond van die Illumina heel-genoom volgordebepaling het alle isolate veelvuldige weerstandigheidsgene bevat, asook IncF plasmied replikons, individueel of in kombinasie met addisionele plasmied tipes. Die IncF replikons en weerstandigheidsgene was geskei; daarom kon assosiasies tussen verskillende IncF replikons en weerstandigheidsgene nie bepaal word nie. Nanopore volgordebepaling het plasmiedontleding van verskeie van die E. coli en K. pneumoniae isolate moontlik gemaak, maar chromosomale gene kon nie gevisualiseer word nie en wansamestellings het tot gefragmenteerde plasmiede aanleiding gegee. Die gebruik van die de novo hibriedsamesteller Unicycler het volle resolusie van alle plasmiede en chromosomale gene in verskeie van die E. coli en K. pneumoniae isolate moontlik gemaak. Onder die E. coli isolate, is drie F-tipe multi-replikon plasmiede en twee enkel-replikon plasmiede, IncI1-y en IncB/O/K/Z, beskryf, wat weerstandigheidsgene gedra het. ‘n Nuwe multi-replikon plasmied, FII(FIC)-FIB-X, is geïdentifiseer, wat die verlengde-spektrum β-laktamase blaTEM-135 en die plasmied-gemedieërde kinoloon weerstandigheidsgeen qnrS1 gedra het. Daar is bevestig dat die blaCTX-M gene op die chromosoom geleë was in drie E. coli isolate, en op F-tipe plasmiede in twee E. coli isolate. Daar is enkel-replikon F-tipe plasmiede, multi-replikon FIB-HI1B saamgestelde plasmiede, en ‘n enkel-replikon IncC plasmied in K. pneumoniae isolate geïdentifiseer. Die FIB-HI1B plasmiede is met blaCTX-M-15, aac(6’)-Ib-cr, en qnrS1 geassosieër. Die blaCTX-M-15 was plasmied-gemedieërd in alle K. pneumoniae isolate. Gevolgtrekking Daar is vasgestel dat die verlengde-spektrum β-laktamase en plasmied-gemedieërde kinoloon weerstandigheidsgene in E. coli isolate gesamentlik op dieselfde plasmiede, asook op aparte plasmiede voorgekom het. In teenstelling hiermee is verlengde-spektrum β-laktamase en plasmied-gemedieërde kinoloon weerstandigheidsgene gesamentlik op dieselfde plasmiede in K. pneumoniae isolate aangetref, en die F-tipe plasmiede het verskil van dié in E. coli isolate. Aangesien slegs twee gemeenskapsverworwe K. pneumoniae isolate Nanopore volgordebepaling ondergaan het, kon gevolgtrekkings oor die verspreiding van K. pneumoniae plasmiede in gesondheidsinstellings en gemeenskappe nie gemaak word nie. Plasmiede van dieselfde FAB-tipes is in E. coli isolate van verskeie volgorde-tipes, en in beide gesondheidsinstellings en gemeenskappe geïdentifiseer, wat verspreiding tussen hierdie gebiede aandui.Master

Feeding Preferences of Omnivorous Gizzard Shad as Influenced by Fish Size and Zooplankton Density

In Ohio reservoirs, larval gizzard shad Dorosoma cepedianum less than 30 mm total length consume only zooplankton but frequently switch to detritus as they grow longer than 30 mm. However, in laboratory studies without detritus, gizzard shad longer than 30 mm consume crustacean zooplankton. To explore the composition of diets of omnivorous 30-100-mm gizzard shad, we completed 1-h laboratory feeding trials with different amounts of zooplankton and detritus and quantified the diets of gizzard shad in reservoirs. In both laboratory and field, gizzard shad ate primarily detritus but also ate zooplankton, consuming more as more became available, which demonstrates that this species is a facultative detritivore. In the field, zooplankton consumption declined as gizzard shad body size increased. We believe gizzard shad maximize growth by supplementing their low-protein detritus diet with more zooplankton as more becomes available. With this strategy, omnivorous gizzard shad may compromise the potential for food web manipulations based on the trophic cascade hypothesis in Ohio reservoirs.This work was supported by Federal Aid in Sport Fish Restoration project F-57-R, administered jointly by the U.S. Fish and Wildlife Service and the Ohio Division of Wildlife, to R.A.S

Comparison of Nitrogen Oxide Metabolism among Diverse Ammonia-Oxidizing Bacteria

Ammonia-oxidizing bacteria (AOB) have well characterized genes that encode and express nitrite reductases (NIR) and nitric oxide reductases (NOR). However, the connection between presence or absence of these and other genes for nitrogen transformations with the physiological production of nitric oxide (NO) and nitrous oxide (N2O) has not been tested across AOB isolated from various trophic states, with diverse phylogeny, and with closed genomes. It is therefore unclear if genomic content for nitrogen oxide metabolism is predictive of net N2O production. Instantaneous microrespirometry experiments were utilized to measure NO and N2O emitted by AOB during active oxidation of ammonia (NH3) or hydroxylamine (NH2OH) and through a period of anoxia. This data was used in concert with genomic content and phylogeny to assess whether taxonomic factors were predictive of nitrogen oxide metabolism. Results showed that two oligotrophic AOB strains lacking annotated NOR-encoding genes released large quantities of NO and produced N2O abiologically at the onset of anoxia following NH3-oxidation. Furthermore, high concentrations of N2O were measured during active O2-dependent NH2OH oxidation by the two oligotrophic AOB in contrast to non-oligotrophic strains that only produced N2O at the onset of anoxia. Therefore, complete nitrifier denitrification did not occur in the two oligotrophic strains, but did occur in meso- and eutrophic strains, even in Nitrosomonas communis Nm2 that lacks an annotated NIR-encoding gene. Regardless of mechanism, all AOB strains produced measureable N2O under tested conditions. This work further confirms that AOB require NOR activity to enzymatically reduce NO to N2O in the nitrifier denitrification pathway, and also that abiotic reactions play an important role in N2O formation, in oligotrophic AOB lacking NOR activity