Clinical Talaromyces marneffei isolates exhibit enhanced stress tolerance and diminished antifungal responsiveness relative to non-clinical strains

Among the 12 species of the genus Yersinia, Yersinia enterocolitica is the most prevalent cause of illness in humans and animals. Among other characteristics, its patogenicity is related to six biotypes: 1B and 2 to 5 considered pathogenic and the 1A biotype considered non-pathogenic. Despite being defined as non-pathogenic, literature has been shown that biotype 1A strains may be the etiological agents of infections in humans and animals. The aim of this work was to investigate the pathogenic potential and to verify the genomic similarity of Y. enterocolitica biotype 1A isolated from clinical and non-clinical sources. Fifty-two strains of Y. enterocolitica biotype 1A isolated from humans (11), animals (11), food (15), and environment (15) were analyzed regarding their susceptibility to antimicrobials, behavior against phenotypic tests related to virulence, resistance to oxygen intermediate reactives, invasion to HEp-2 and Caco-2 cells, presence of virulence genes by PCR, and genomic similarity by Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) and Pulsed-field gel electrophoresis (PFGE). Both clinical and non-clinical strains showed resistance to ampicillin and cephalothin. It was not observed any difference in the pathogenic potential between clinical and non-clinical strains face of the following tests: salicine fermentation, esculin hydrolysis, pirazinamidase activity, oxygen intermediate reactives and HEp-2 cell invasion assay. On the other hand, the non-clinical strains were more invasive to Caco-2 cells than the clinical ones. Eight of 11 studied virulence genes were found. Genes ystB, hreP and fepD were more often detected in clinical strains. In contrast, myfA, fepA, fes and tccC were presented more frequently in non-clinical strains. However, the frequency difference of those genes was not statistically significant between clinical and non-clinical strains. The inv gene was detected in all the strains studied; but no ail, ystA, and virF genes were found in any of the 52 strains. ERIC-PCR and PFGE dendogram allowed the visualization of two groups named A and B. It was observed a high genomic similarity among almost all human and animal isolated strains (>63%), as well as a high genomic similarity between the clinical and non-clinical strains (>58%). The discriminatory index for ERIC-PCR was 0.98 and for PFGE was 0.99. Among biotype 1A strains no difference was observed between the pathogenic potential of clinical and non-clinical strains face to the phenotype tests employed, and regarding the prevalence of the studied virulence genes. The exception was the Caco-2 cells invasion assay where non-clinical strains were more invasive., ERIC-PCR and PFGE discriminated the studied strains similarly. The high genomic similarity between the clinical and non-clinical strains gives evidence that animals constitute important reservoirs of Y.enterocolitica biotype 1A and suggests that environmental and food isolates have been the source of human and animal infections.

The anaerobic bacterium Clostridioides difficile, renamed from Clostridium difficile, substantially accounts for nosocomial, antibiotic-associated infections worldwide. The complex dynamics of various traits shape the bacterial virulence, such as toxin production and gene sequence variants, multidrug resistances, and sporulation. C. difficile is also known for its mobile genome that comprises diverse mobile genetic elements (MGE), thereby enabling the spread of advantageous genes. The pathogen is extensively investigated to enlighten its virulence and dissemination. Strain isolation is mainly done in clinical context, although C. difficile also inhabits human and mammalian intestines without disease manifestation or is present in environmental surroundings. Further, these isolations always employ antibiotic based cultivation, which might lead to an isolation bias. Within this thesis, non-clinical strains were isolated from environmental samples. Five strains in total were obtained from which two were isolated without antibiotic treatment. An effective protocol for antibiotic-free isolation could not be established but offers potential for further efforts. Isolation approaches were supported by the established detection PCR that targets the C. difficile hpdBCA-operon and detects even low abundances of C. difficile in environmental DNA. Specificity of the detection PCR was verified by next generation sequencing of detection PCR amplicons from environmental samples to ensure that amplified sequences only originated from C. difficile DNA. These environmental samples were also analyzed via 16S rRNA gene sequencing, in which C. difficile could not be detected, what clearly demonstrated their low abundance. Besides validation of environmental sources for promising C. difficile isolation, amplicons from the established detection PCR further allow phylogenetic estimation, which was determined by average nucleotide identity analyses of representative sequences. Analysis of PCR amplicons also supported the assumed isolation bias due to antibiotic treatment, as detected sequences from environmental samples did not accord with sequences from strains obtained by antibiotic-based protocols. This indicated an abundance shift of the present C. difficile strains and selective enrichment of the un-detected strains upon antibiotic treatment. The genomes of the non-clinical strains isolated within this thesis were compared to clinical reference strains to identify clinical-related features with influence on strain virulence. Besides analysis of crucial virulence factors, the genome analyses addressed MGEs, and further included antibiotic resistances and accessory genes, which were all incorporated in pairwise genome alignments. An overall trend of more ARGs, MGEs and accessory genes in the clinical strains was observed. The accessory genes mainly comprised specific functions such as transcription, which are correlated to higher strain virulence. This observation might further indicate the ability of the clinical strains to adapt more rapidly to environmental fluctuations. It is therefore advisable to increasingly implement non-clinical strains in comparative analyses to unravel C. difficile virulence progression. ARG, MGE and pan genome analyses were put into genomic context to identify conjunctions between specific elements. This revealed that accessory genes, which were identified to correlate with higher strain virulence, resided within MGEs. This connection highlights the relevance of MGEs in C. difficile virulence. All non-clinical isolates and the clinical reference strains were subjected to phenotypic investigations on spontaneous and induced prophage activity. Active prophages were determined by isolation and sequencing of particle-protected DNA and mapping of sequencing reads onto the host genomes. Phages were either not induced, or induced with the secondary bile salt deoxycholate, which is a stressful component in the natural C. difficile habitat. Corresponding clinical and non-clinical strains showed no difference in prophage carriage or tolerance to deoxycholate, as determined by relative growth in the presence of varying concentrations. The sequencing data verified spontaneous prophage activity in all strains with one exception and revealed multiple active phages co-existing within the same host. The inducing effect of deoxycholate was also confirmed and observed for almost all phages, which underlines the relevance of natural conditions in investigations of C. difficile phage activity. Differences in increased phage activity upon induction could be observed between corresponding strains, but the data could not ensure a clinical relation. One corresponding strain pair however indicated a connection between the higher induction in the clinical strain and its higher reactivity determined in the comprehensive genome analyses. Fourteen novel phages in total were identified. Further, several regions with clear activity but missing phage identity were apparent in the sequencing read mapping. Different mechanisms might be responsible for the envelopment of these mobile DNA elements, such as phage-mediated transduction. One of these active DNA elements could be assigned to lateral transduction, which is the first description of this mechanism in C. difficile. These findings point to so far unraveled HGT strategies in C. difficile and encourage further investigations.

Certain strains of Enterococcus faecium and Enterococcus faecalis contribute beneficially to animal health and food production, while others are associated with nosocomial infections. To determine whether there are structural and functional genomic features that are distinct between nonclinical (NC) and clinical (CL) strains of those species, we analyzed the genomes of 31 E. faecium and 38 E. faecalis strains. Hierarchical clustering of 7,017 orthologs found in the E. faecium pangenome revealed that NC strains clustered into two clades and are distinct from CL strains. NC E. faecium genomes are significantly smaller than CL genomes, and this difference was partly explained by significantly fewer mobile genetic elements (ME), virulence factors (VF), and antibiotic resistance (AR) genes. E. faecium ortholog comparisons identified 68 and 153 genes that are enriched for NC and CL strains, respectively. Proximity analysis showed that CL-enriched loci, and not NC-enriched loci, are more frequently colocalized on the genome with ME. In CL genomes, AR genes are also colocalized with ME, and VF are more frequently associated with CL-enriched loci. Genes in 23 functional groups are also differentially enriched between NC and CL E. faecium genomes. In contrast, differences were not observed between NC and CL E. faecalis genomes despite their having larger genomes than E. faecium. Our findings show that unlike E. faecalis, NC and CL E. faecium strains are equipped with distinct structural and functional genomic features indicative of adaptation to different environments.

The pathogenic bacterium Clostridioides difficile is a worldwide health burden with increasing morbidity, mortality and antibiotic resistances. Therefore, extensive research efforts are made to unravel its virulence and dissemination. One crucial aspect for C. difficile is its mobilome, which for instance allows the spread of antibiotic resistance genes (ARG) or influence strain virulence. As a nosocomial pathogen, the majority of strains analyzed originated from clinical environments and infected individuals. Nevertheless, C. difficile can also be present in human intestines without disease development or occur in diverse environmental habitats such as puddle water and soil, from which several strains could already be isolated. We therefore performed comprehensive genome comparisons of closely related clinical and non-clinical strains to identify the effects of the clinical background. Analyses included the prediction of virulence factors, ARGs, mobile genetic elements (MGEs), and detailed examinations of the pan genome. Clinical-related trends were thereby observed. While no significant differences were identified in fundamental C. difficile virulence factors, the clinical strains carried more ARGs and MGEs, and possessed a larger accessory genome. Detailed inspection of accessory genes revealed higher abundance of genes with unknown function, transcription-associated, or recombination-related activity. Accessory genes of these functions were already highlighted in other studies in association with higher strain virulence. This specific trend might allow the strains to react more efficiently on changing environmental conditions in the human host such as emerging stress factors, and potentially increase strain survival, colonization, and strain virulence. These findings indicated an adaptation of the strains to the clinical environment. Further, implementation of the analysis results in pairwise genome comparisons revealed that the majority of these accessory genes were encoded on predicted MGEs, shedding further light on the mobile genome of C. difficile. We therefore encourage the inclusion of non-clinical strains in comparative analyses.

The rare occurrence of human cryptococcosis caused by Cryptococcus gattii sensu lato leads to difficulties in establishing the antifungal susceptibility profile between species of this potentially lethal pathogen, which may be crucial for treating cryptococcosis. To establish an antifungal susceptibility profile of C. gattii s.l. in Taiwan. A total of 104 environmental C. gattii s.l. strains (including multilocal sequence typing ST7, ST106, ST274, ST328, ST546, ST548 and ST630) and 21 previously collected clinical strains (including ST7, ST44, ST06, ST274, ST328 and ST329) were included in this study. We determined the minimum inhibitory concentrations (MICs) of six antifungal agents (itraconazole, fluconazole, voriconazole, posaconazole, flucytosine and amphotericin B) against environmental C. gattii s.l. strains and compared the antifungal susceptibility profiles of environmental strains with those of clinical strains. The antifungal susceptibility data demonstrated that the MICs of antifungal agents against environmental strains were comparable to those against clinical strains. Compared with strains of Cryptococcus deuterogattii, those of C. gattii sensu stricto were more susceptible to azoles and flucytosine. The differences in antifungal susceptibility between the strains of each sequence type (ST) were significant. Correlation analysis of MICs revealed cross-resistance between azoles in environmental strains of C. gattii s.l. Geographic differences in the antifungal susceptibility of C. gattii s.l. isolated from different cities in Taiwan were observed in this study. Clinical and environmental strains were indistinguishable in antifungal susceptibility. The antifungal susceptibility of C. gattii s.l. is associated with STs. Therefore, establishing an ST-oriented domestic antifungal susceptibility database may help treat C. gattii s.l.-induced cryptococcosis.

Modulation of the membrane permeability through a decrease in porin-mediated antibiotic entry and/or an increase in antibiotic efflux is one of the resistance mechanisms to antibiotics evolved by Gram-negative bacteria. To assess whether the outer membrane porin OprD and Resistance-Nodulation-Division (RND) efflux pumps were similarly expressed in 33 ciprofloxacin-resistant clinical strains of Pseudomonas aeruginosa and in 30 non-clinical strains originating from the hospital environment (mainly waterborne Pseudomonas aeruginosa), the expression of oprD, mexB, mexF, and mexY genes was investigated. Overall, the expression of oprD was not detected by RT-qPCR in 14 (22%) strains and underexpressed in 35 (56%) more. No significant difference in oprD expression was detected between clinical and non-clinical strains. As for efflux pumps, 23 (70%) of the clinical strains overexpressed at least one of the tested RND genes. Overexpression of mexB, mexF and mexY was detected in 27, 12, and 45% of the clinical strains, respectively. In the 30 non-clinical strains, no overexpression could be found for mexB, mexF, or mexY. On the contrary, a global underexpression of the tested efflux pump genes was recorded. In both clinical and environmental strains, a positive correlation was found between the expressions of oprD and mexB. Similarly, the expressions of oprD and mexF were positively correlated. This result contrasts with the inverse correlation between both MexAB-OprM/MexEF-OprN and OprD previously described in carbapenem-resistant P. aeruginosa strains. The only positive correlation between phenotypic ciprofloxacin minimum inhibitory concentrations (MICs) and the expression of efflux pump gene was witnessed with mexY (analysis on pooled results for clinical and environmental strains). However, in clinical strains, no statistically significant link could be found between the degree of reduction in ciprofloxacin MICs witnessed with Phenylalanine-Arginine β-naphthylamide (PAβN) and the expression of any of the 3 RND genes tested.

Molecular Characterization of Clinical Saccharomyces cerevisiae Isolates and their Association with Non-Clinical Strains

Clinical strains of Mycobacterium avium isolated from patients with acquired immunodeficiency syndrome, but not a non-clinical laboratory strain (ATCC 25291), were found to stimulate the human alveolar epithelial cell line A549, to produce monocyte chemoattractant protein (MCP)-1. A549 cells were also found to produce elevated levels of MCP-1 in response to sonicates of the clinical strains of M. avium, and surprisingly, the non-clinical strain as well. However, sonic extracts of the clinical strains were found to induce significantly higher levels of MCP-1 production compared to extracts of the non-clinical strain (P < 0.001). These data suggest the existence of strain-related differences in antigen expression by M. avium. The clinical and non-clinical strains of M. avium were found to attach and invade, but not replicate in A549 cells indicating that MCP-1 production by A549 cells does require the presence of viable, replicating organisms. Activation of alveolar epithelial cells by exposure to M. avium resulting in the production of chemokines which recruit inflammatory cells to the site of infection may be an important regulatory pathway for the activation of pulmonary host defense.

Clinical strains of Mycobacterium avium isolated from patients with acquired immunodeficiency syndrome, but not a non-clinical laboratory strain (ATCC 25291), were found to stimulate the human alveolar epithelial cell line A549, to produce monocyte chemoattractant protein (MCP)-1. A549 cells were also found to produce elevated levels of MCP-1 in response to sonicates of the clinical strains of M. avium, and surprisingly, the non-clinical strain as well. However, sonic extracts of the clinical strains were found to induce significantly higher levels of MCP-1 production compared to extracts of the non-clinical strain ( P<0.001). These data suggest the existence of strain-related differences in antigen expression by M. avium. The clinical and non-clinical strains of M. avium were found to attach and invade, but not replicate in A549 cells indicating that MCP-1 production by A549 cells does require the presence of viable, replicating organisms. Activation of alveolar epithelial cells by exposure to M. avium resulting in the production of chemokines which recruit inflammatory cells to the site of infection may be an important regulatory pathway for the activation of pulmonary host defense.

In this study, a number of Listeria monocytogenes strains of different origins were evaluated for in vitro invasion capacity for various human cell types (monocytic THP-1, enterocytic Caco-2, and hepatocytic HepG2 cells) and for expression levels of specific virulence genes. For THP-1 cells, no differences between clinical and nonclinical L. monocytogenes strains in invasion capacity or in production of the proinflammatory cytokine interleukin-8 (IL-8) were observed, whereas for the Caco-2 and HepG2 cells, significant differences in invasion capacity were noticed. On average, the clinical strains showed a significantly lower invasion capacity than the nonclinical L. monocytogenes strains. Furthermore, it was shown that the clinical strains induce lower IL-8 levels in HepG2 cells than do the nonclinical strains. This observation led us to study the mRNA expression levels of inlA, inlB, and ami, important virulence genes mediating adhesion and invasion of eukaryotic cells, by real-time reverse transcription-PCR for 27 clinical and 37 nonclinical L. monocytogenes strains. Significant differences in inlA and inlB expression were observed, with clinical strains showing a lower expression level than nonclinical strains. These observations were in accordance with in vitro invasion of Caco-2 and HepG2 cells, respectively. The results of this study indicate that differential expression levels of inlA and inlB possibly play a role in the virulence capacities of L. monocytogenes strains. The lower capacity of clinical strains to invade HepG2 cells and to induce IL-8 is possibly a mechanism of immune evasion used by specific L. monocytogenes strains.

The understanding of the molecular basis of the flux of antimicrobial resistance genes (ARG) is a fundamental requirement to develop a comprehensive explanation of how pathogenic strains adapt to extreme resistance phenotypes at a global scale. However, it is difficult to assess how ARG interact with human activities. This constraint led us to seek a suitable biological model system, which has two components: class 1 integrons as the main molecular element of bacterial resistance to antibiotics, and Tierra del Fuego Island as the study area. Both clinical and non-clinical strains were studied from this island for the presence of class 1 integrons. The 30 % of clinical strains and the 11 % of strains isolated from the open environment were intI1 positive, respectively, sharing 3 intI1 “clinical” alleles previously described in nosocomial strains from Europe, Africa, and Asia, depicting an interchange of genes among both habitats, the hospital and the sites close to human activities in Ushuaia City. It is likely that once the intI1-positive clinical strains are released from the hospital, the intI1 genes can be transferred through the mechanisms of lateral genetic transfer to other bacterial species from the open environment where they can be maintained over time.

Burkholderia cepacia is found in soils and waters, it can be used in biocontrol and bioremediation but is also a human pathogen. It is not yet clear what differentiates pathogenic from non-pathogenic strains of the organism. In this study the multiple replicon structure was investigated in 28 strains of B. cepacia by pulsed field gel electrophoresis. All strains examined, whether of clinical, environmental or plant pathogenic origin, were found to have two, three or four large (> 500 kbp) replicons. Many strains also contained small replicons. Clinical strains were more likely to have three or four large replicons than non-clinical strains. Multiple replicon structure was also demonstrated in B. gladioli and Alcaligenes eutrophus.

Acinetobacter baumannii, an important pathogen known for its widespread antibiotic resistance, has been the focus of extensive research within its genus, primarily involving clinical isolates. Consequently, data on environmental A. baumannii and other Acinetobacter species remain limited. Here, we utilized Illumina and Nanopore sequencing to analyze the genomes of 10 Acinetobacter isolates representing 6 different species sourced from aquatic environments in South Australia. All 10 isolates were phylogenetically distinct compared to clinical and other non-clinical Acinetobacter strains, often tens of thousands of single-nucleotide polymorphisms from their nearest neighbors. Despite the genetic divergence, we identified pdif modules (sections of mobilized DNA) carrying clinically important antimicrobial resistance genes in species other than A. baumannii, including carbapenemase oxa58, tetracycline resistance gene tet(39), and macrolide resistance genes msr(E)-mph(E). These pdif modules were located on plasmids with high sequence identity to those circulating in globally distributed A. baumannii ST1 and ST2 clones. The environmental A. baumannii isolate characterized here (SAAb472; ST350) did not possess any native plasmids; however, it could capture two clinically important plasmids (pRAY and pACICU2) with high transfer frequencies. Furthermore, A. baumannii SAAb472 possessed virulence genes and a capsular polysaccharide type analogous to clinical strains. Our findings highlight the potential for environmental Acinetobacter species to acquire and disseminate clinically important antimicrobial resistance genes, underscoring the need for further research into the ecology and evolution of this important genus.IMPORTANCEAntimicrobial resistance (AMR) is a global threat to human, animal, and environmental health. Studying AMR in environmental bacteria is crucial to understand the emergence and dissemination of resistance genes and pathogens, and to identify potential reservoirs and transmission routes. This study provides novel insights into the genomic diversity and AMR potential of environmental Acinetobacter species. By comparing the genomes of aquatic Acinetobacter isolates with clinical and non-clinical strains, we revealed that they are highly divergent yet carry pdif modules that encode resistance to antibiotics commonly used in clinical settings. We also demonstrated that an environmental A. baumannii isolate can acquire clinically relevant plasmids and carries virulence factors similar to those of hospital-associated strains. These findings suggest that environmental Acinetobacter species may serve as reservoirs and vectors of clinically important genes. Consequently, further research is warranted to comprehensively understand the ecology and evolution of this genus.

Yersinia enterocolitica biotype 1A (B1A) strains are considered as non-pathogenic; however, some reports have identified some strains as the causal agents of infection. In South America, few studies molecularly characterized the strains of this biotype. This work typed 51 B1A strains isolated from clinical and non-clinical sources from Brazil and Chile by Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) to elucidate their genotypic diversity, and verify the distribution of 11 virulence markers by PCR. The strains were divided into two groups, ERIC-A and ERIC-B, clustered independently of their clinical or non-clinical origin. No differences were observed in the frequencies of the virulence markers between clinical and non-clinical strains. However, the genes ystB, hreP and myfA occurred exclusively in the strains of the group ERIC-A. Some clinical and non-clinical strains were clustered in the same genetic group and presented the same number of virulence markers, which might suggest the role of the environment and food as a potential source of infection for humans and animals. The results corroborate with the hypothesis that B1A strains are divided into two main clusters that differ in the frequency of some virulence markers, a fact observed for the first time in South American strains.

It is well documented that disturbance of cell surface by some agents triggers compensatory responses aimed to maintain the cell wall integrity in fungi and other organisms. Here, the thermodimorphic fungus Sporothrix globosa, a member of the pathogenic clade of the Sporothrix complex, was propagated in yeast-peptone-dextrose medium under conditions to obtain the mycelium (pH 4.5, 27-28°C) or the yeast (pH 7.8, 32-34°C) morphotypes in the absence and presence of the wall-interacting dyes Congo Red (CR) and Calcofluor White (CFW) either alone or in combination. After different periods of time, growth, cell morphology and activity of glucosamine-6-phosphate synthase (GlcN-6-P synthase), an ubiquitous enzyme that plays a crucial role in cell wall biogenesis, were determined. CR and to a lower extent CFW affected growth and morphology of both fungal morphotypes and significantly increased enzyme activity. Notoriously, CR or CR in combination with CFW induced the transient conversion of yeasts into conidia-forming filamentous cells even under culture conditions adjusted for yeast development, most likely as a strategy to evade the noxious effect of the dye. After sometime, hypha returned to yeast cells. An hypothetical model to explain the effect of CR on morphology and enzyme activity based on the possible role of membrane-spanning proteins known as mechanosensors is proposed. Results are discussed in terms of the fungal responses to cell wall damage.