Tellurite Resistance Research Articles

Antimicrobial efficacy and amino acid substitutions associated with susceptibility to the tellurium compound AS101 against Haemophilus influenzae and Haemophilus parainfluenzae.

The tellurite toxicity in Haemophilus influenzae and H. parainfluenzae remains unclear. To understand the potential of tellurite as a therapeutic option for these bacteria, we investigated the antimicrobial efficacy of AS101, a tellurium compound, against H. influenzae and H. parainfluenzae and the molecular basis of their differences in AS101 susceptibility. Through broth microdilution, we examined the minimum inhibitory concentration (MIC) of AS101 in 51 H. influenzae and 28 H. parainfluenzae isolates. Whole-genome sequencing was performed on the H. influenzae isolates to identify genetic variations associated with AS101 susceptibility. The MICs of AS101 were ≦ 4, 16-32, and ≧ 64μg/mL in 9 (17.6%), 12 (23.5%), and 30 (58.8%) H. influenzae isolates, respectively, whereas ≦ 0.5μg/mL in all H. parainfluenzae isolates, including multidrug-resistant isolates. Time-killing kinetic assay and scanning electron microscopy revealed the in vitro bactericidal activity of AS101 against H. parainfluenzae. Forty variations in nine tellurite resistance-related genes were associated with AS101 susceptibility. Logistic regression, receiver operator characteristic curve analysis, Venn diagram, and protein sequence alignment indicated that Val195Ile substitution in TerC, Ser93Gly in Gor (glutathione reductase), Pro44Ala/Ala50Pro in NapB (nitrate reductase), Val307Leu in TehA (tellurite resistance protein), Cys105Arg in CysK (cysteine synthase), and Thr364Ser in Csd (Cysteine desulfurase) were strongly associated with reduced AS101 susceptibility, whereas Ser155Pro in TehA with increased AS101 susceptibility. In conclusions, the antimicrobial efficacy of AS101 is high against H. parainfluenzae but low against H. influenzae. Genetic variations and corresponding protein changes relevant to AS101 non-susceptibility in H. influenzae were identified.

Novel phages of Pseudomonas syringae unveil numerous potential auxiliary metabolic genes.

Relatively few phages that infect plant pathogens have been isolated and investigated. The Pseudomonas syringae species complex is present in various environments, including plants. It can cause major crop diseases, such as bacterial canker on apricot trees. This study presents a collection of 25 unique phage genomes that infect P. syringae. These phages were isolated from apricot orchards with bacterial canker symptoms after enrichment with 21 strains of P. syringae. This collection comprises mostly virulent phages, with only three being temperate. They belong to 14 genera, 11 of which are newly discovered, and 18 new species, revealing great genetic diversity within this collection. Novel DNA packaging systems have been identified bioinformatically in one of the new phage species, but experimental confirmation is required to define the precise mechanism. Additionally, many phage genomes contain numerous potential auxiliary metabolic genes with diversified putative functions. At least three phages encode genes involved in bacterial tellurite resistance, a toxic metalloid. This suggests that viruses could play a role in bacterial stress tolerance. This research emphasizes the significance of continuing the search for new phages in the agricultural ecosystem to unravel novel ecological diversity and new gene functions. This work contributes to the foundation for future fundamental and applied research on phages infecting phytopathogenic bacteria.

Increased resistance against tellurite is conferred by a mutation in the promoter region of uncommon tellurite resistance gene tehB in the ter-negative Shiga toxin-producing Escherichia coli O157:H7.

Selective agents are important for isolating Shiga toxin-producing Escherichia coli (STEC) because the undesirable growth of microflora should be inhibited. Potassium tellurite (PT) is a common selective agent for major STEC serotypes. In this study, we found a novel variant of PT resistance genes, tehAB, in STEC O157:H7. Molecular experiments clearly showed that one point mutation in a predicted internal promoter region of tehB upregulated the expression of the gene and consequently led to increased resistance to PT. Because tehAB genes are ubiquitous across E. coli, these results provide universal insight into PT resistance in this species.

Characteristics and phylogenetic distribution of megaplasmids and prediction of a putative chromid in Pseudomonas aeruginosa

Research on megaplasmids that contribute to the spread of antimicrobial resistance (AMR) in Pseudomonas aeruginosa strains has grown in recent years due to the now widely used technologies allowing long-read sequencing. Here, we systematically analyzed distinct and consistent genetic characteristics of megaplasmids found in P. aeruginosa. Our data provide information on their phylogenetic distribution and hypotheses tracing the potential evolutionary paths of megaplasmids. Most of the megaplasmids we found belong to the IncP-2-type, with conserved and syntenic genetic backbones carrying modules of genes associated with chemotaxis apparatus, tellurite resistance and plasmid replication, segregation, and transmission. Extensively variable regions harbor abundant AMR genes, especially those encoding β-lactamases such as VIM-2, IMP-45, and KPC variants, which are high-risk elements in nosocomial infection. IncP-2 megaplasmids act as effective vehicles transmitting AMR genes to diverse regions. One evolutionary model of the origin of megaplasmids claims that chromids can develop from megaplasmids. These chromids have been characterized as an intermediate between a megaplasmid and a chromosome, also containing core genes that can be found on the chromosome but not on the megaplasmid. Using in silico prediction, we identified the “PABCH45 unnamed replicon” as a putative chromid in P. aeruginosa, which shows a much higher similarity and closer phylogenetic relationship to chromosomes than to megaplasmids while also encoding plasmid-like partition genes. We propose that such a chromid could facilitate genome expansion, allowing for more rapid adaptations to novel ecological niches or selective conditions, in comparison to megaplasmids.

Unexpected role of pig nostrils in the clonal and plasmidic dissemination of extended-spectrum beta-lactamase-producing Escherichia coli at farm level

The presence of methicillin-resistant or -susceptible S. aureus in pig nostrils has been known for a long time, but the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli has hardly been investigated. Here, we collected 25 E. coli recovered from nasal samples of 40 pigs/10 farmers of four farms. Nine ESBL-producing isolates belonging to ST48, ST117, ST847, ST5440, ST14914 and ST10 were retrieved from seven pigs. All blaESBL genes (blaCTX-M-32,blaCTX-M-14,blaCTX-M-1,blaCTX-M-65, and blaSHV-12) were horizontally transferable by conjugation through plasmids belonging to IncI1 (n=3), IncX1 (n=3) and IncHI2 (n=1) types. IncI1-plasmids displayed different genetic environments: i) IS26-blaSHV-12-deoR-IS26, ii) wbuC-blaCTX-M-32-ISKpn26 (IS5), and iii) IS930-blaCTX-M-14-IS26. The IncHI2-plasmid contained the genetic environment IS903-blaCTX-M-65-fipA with multiple resistance genes associated either to: a) Tn21-like transposon harbouring genes conferring aminoglycosides/beta-lactams/chloramphenicol/macrolides resistance located on two atypical class 1 integrons with an embedded ΔTn5393; or b) Tn1721-derived transposon displaying an atypical class 1 integron harbouring aadA2-arr3-cmlA5-blaOXA-10-aadA24-dfrA14, preceding the genetic platform IS26-blaTEM-95-tet(A)-lysR-floR-virD2-ISVsa3-IS3075-IS26-qnrS1, as well as the tellurite resistance module. Other plasmids harbouring clinically relevant genes were detected, such as a ColE-type plasmid carrying the mcr-4.5 gene. Chromosomally encoded genes (fosA7) or integrons (intI1-dfrA1-aadA1-qacE-sul1/intI1-IS15-dfrA1-aadA2) were also identified. Finally, an IncY plasmid harbouring a class 2 integron (intI2-dfrA1-sat2-aadA1-qacL-IS406-sul3) was detected but not associated with a blaESBL gene. Our results evidence that pig nostrils might favour the spread of ESBL-E. coli and mcr-mediated colistin-resistance. Therefore, enhanced monitoring should be considered, especially in a sector where close contact between animals in intensive farming increases the risk of spreading antimicrobial resistance.

The tellurite resistance gene cluster of pathogenic bacteria and its effect on oxidative stress response.

Tellurite resistance gene clusters have been identified in numerous pathogenic bacteria, including clinical isolates of Escherichia coli. The rareness of tellurium in host organisms and the noncontaminated environment raises a question about the true functionality of tellurite resistance gene clusters in pathogenesis and their possible contribution to bacterial fitness. The study aims to point out the beneficial effects of the tellurite resistance gene cluster of pathogenic bacteria to survive in ROS-rich environments. Here, we analysed the bacterial response to oxidative stress conditions with and without tellurite resistance gene clusters, which are composed of terWY1XY2Y3 and terZABCDEF genes. By measuring the levels of protein carbonylation, lipid peroxidation, and expression changes of oxidative stress genes upon oxidative stress, we propose a tellurite resistance gene cluster contribution to the elimination of oxidative damage, potentially increasing fitness and resistance to reactive oxygen species during macrophage attack. We have shown a different beneficial effect of various truncated versions of the tellurite resistance gene cluster on cell survival. The terBCDEF genes increased the survival of E. coli strain MC4100 by 13.21%, terW and terZABCDEF by 10.09%, and terWY1XY2Y3 and terZABCDEF by 25.57%, respectively. The ability to survive tellurite treatment is the most significant at 44.8% in wild clinical strain KL53 compared to laboratory strain E. coli MC4100 due to a complete wild-type plasmid presence.

Phenotypic and resistome analysis of antibiotic and heavy metal resistance in the Antarctic bacterium Pseudomonas sp. AU10.

Resistance to antibiotics and heavy metals in Antarctic bacteria has been investigated due to anthropogenic impact on the continent. However, there is still much to learn about the genetic determinants of resistance in native bacteria. In this study, we investigated antibiotic, heavy metal, and metalloid resistance in Pseudomonas sp. AU10, isolated from King George Island (Antarctica), and analyzed its genome to look for all the associated genetic determinants (resistome). We found that AU10 displayed resistance to Cr(VI), Cu(II), Mn(II), Fe(II), and As(V), and produced an exopolysaccharide with high Cr(VI)-biosorption capacity. Additionaly, the strain showed resistance to aminopenicillins, cefotaxime, aztreonam, azithromycin, and intermediate resistance to chloramphenicol. Regarding the resistome, we did not find resistance genes in AU10's natural plasmid or in a prophage context. Only a copper resistance cluster indicated possible horizontal acquisition. The mechanisms of resistance found were mostly efflux systems, several sequestering proteins, and a few enzymes, such as an AmpC β-lactamase or a chromate reductase, which would account for the observed phenotypic profile. In contrast, the presence of a few gene clusters, including the terZABCDE operon for tellurite resistance, did not correlate with the expected phenotype. Despite the observed resistance to multiple antibiotics and heavy metals, the lack of resistance genes within evident mobile genetic elements is suggestive of the preserved nature of AU10's Antarctic habitat. As Pseudomonas species are good bioindicators of human impact in Antarctic environments, we consider that our results could help refine surveillance studies based on monitoring resistances and associated resistomes in these populations.

Genomic Profiling of Multidrug Efflux Pumps and Heavy Metal Proteins in Multidrug-resistant Campylobacter fetus Isolated from Sheath Wash Samples of Bulls in South Africa

A substantial evolution of resistance mechanisms among zoonotic bacteria has resulted from anthropogenic factors related to the application of antibiotics in human and veterinary medicine, particularly in contemporary agriculture. This issue associated with the presence of heavy metal-laced protein in zoonotic bacteria should be taken seriously with regard to the health of animals and the general people. To address this issue, the present study employed whole genome sequencing to identify the antimicrobial resistance patterns of Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv), resistance and virulence genes, as well as heavy metal protein. Based on culture method biochemical testing and PCR amplification using particular primer pairs (MG3F-MG4R and VenSF-VenSR), bacteria were isolated and identified as C. fetus subsp. fetus and C.fetus subsp. venerealis. Subsequently, antimicrobial disc diffusion tests and whole genome sequencing were performed. Isolated bacteria were resistant to tetracycline at 65%, amoxicillin, and doxycycline at 60%. The resistance was also observed against neomycin at (55%), streptomycin (60%), and gentamycin (55%). Through comprehensive genome sequencing analysis and PCR, multiple efflux pumps linked to multidrug resistance were identified, including the broad-specificity multidrug efflux pump (YkkD), along with CmeA, CmeB, CmeC, and gryA.The genome sequence also revealed genes associated with the production of Cytotoxin (Cdt A, B, and C), adhesion and colonization (VirB10 and VirB9), and invasion (CiaB). In addition, different genomic features in heavy metal resistance included Cobalt-zinc-cadmium resistance protein (CzcD), Tellurite resistance protein (TehA), and arsenic efflux pump protein. The findings of the current study revealed that the emergence of bacterial multidrug resistance is increasingly associated with the substantial and growing contribution of Multidrug resistance efflux pumps, as evident in Cff and Cfv. Therefore, it is crucial to tighten the control of Cff and Cfv in livestock production to prevent the transfer of genes resistant to humans through the food chain.

Dissecting the metal resistance genes contributed by virome from mining-affected metal contaminated soils

Virus-borne auxiliary genes have been demonstrated to influence their hosts’ characteristics and flexibility, resulting in an elevated ability to withstand diverse conditions and competition. However, the research on the soil viral community and its influencing factors is not extensive. The effects of bacterium-phage co-evolution in the metal contaminated environment have yet to be fully understood. In our research, numerous viral genes with conserved functional residues/motifs working on detoxifying heavy metals were pinpointed in the virome recovered from 58 contaminated soil metagenomic samples of mineral area, including tellurite resistance genes (e.g., terC, terD), copper resistance genes (e.g., copC, copA), and arsenate resistance genes (e.g., arsC). Moreover, viral proteins involved in key processes of nutrient uptake and metabolism, cellular function, polysaccharides production and biomineralization were also detected, which may also contribute to the response of microbial community to heavy metal stress. Our research showed that viruses have helped their hosts to acquire novel metal-resistance abilities through horizontal gene transfer (HGT) during the adaptation to challenging metal-rich environments. This was also evidenced by the high Codon adaption index (CAI) values across metal-resistance-related genes. This study has advanced our understanding on virus-host interplay within heavy metal contaminated soils.

Global population structure, genomic diversity and carbohydrate fermentation characteristics of clonal complex 119 (CC119), an understudied Shiga toxin-producing E. coli (STEC) lineage including O165:H25 and O172:H25.

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of various serotypes, O157:H7 and five major non-O157 STEC (O26:H11, O111:H8, O103:H2, O121:H19 and O145:H28) can be selectively isolated by using tellurite-containing media. While human infections by O165:H25 STEC strains have been reported worldwide, their detection and isolation are not easy, as they are not resistant to tellurite. Systematic whole-genome sequencing (WGS) analyses have not yet been conducted. Here, we defined O165:H25 strains and their close relatives, including O172:H25 strains, as clonal complex 119 (CC119) and performed a global WGS analysis of the major lineage of CC119, called CC119 sensu stricto (CC119ss), by using 202 CC119ss strains, including 90 strains sequenced in this study. Detailed comparisons of 13 closed genomes, including 7 obtained in this study, and systematic analyses of Stx phage genomes in 50 strains covering the entire CC119ss lineage, were also conducted. These analyses revealed that the Stx2a phage, the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS), many prophages encoding T3SS effectors, and the virulence plasmid were acquired by the common ancestor of CC119ss and have been stably maintained in this lineage, while unusual exchanges of Stx1a and Stx2c phages were found at a single integration site. Although the genome sequences of Stx2a phages were highly conserved, CC119ss strains exhibited notable variation in Stx2 production levels. Further analyses revealed the lack of SpLE1-like elements carrying the tellurite resistance genes in CC119ss and defects in rhamnose, sucrose, salicin and dulcitol fermentation. The genetic backgrounds underlying these defects were also clarified.

Metabolite profiling, in vitro and in silico assessment of antibacterial and anticancer activities of Alternaria alternata endophytic in Jatropha heynei.

Fungal endophytes produce a range of structurally diverse metabolites with bioactive principles. In this study, an endophytic fungus Alternaria alternata was isolated from Jatropha heynei and cultured in potato dextrose liquid broth. Culture filtrate of A. alternata was extracted in ethyl acetate and metabolites were characterized by QTOF-HRLCMS. Among compounds detected, spectral compounds such as kigelinone, and levofuraltadone were reported with antibacterial property, while 2-hydroxychrasophanol, isoathyriol, glycophymoline, columbianetin and kaempferol 3-O-β-D- galactoside were reported with cytotoxic properties. Partially purified metabolites of A. alternata showed significant antibacterial activity against tested clinical bacterial strains by agar well diffusion method. High zone of inhibition was recorded against Enterococcus faecalis, Pseudomonas syringae and Klebsiella pneumoniae. In vitro anticancer activity of fungal extract by MTT assay displayed high cytotoxic effect on human lung carcinoma cancer cell line (A549) with IC50 value of 393.52µgml-1, and without any significant cytotoxic effect on human breast cancer cell line (MCF-7). Further, antibacterial and anticancer spectral compounds of A. alternata were subjected to molecular docking analysis with antibacterial target proteins such as tellurite resistance protein (2JXU), indole-3-acetaldehyde dehydrogenase (5IUU) and alkyl hydroperoxide reductase (5Y63), and anticancer target human apoptotic regulator protein (1G5M). The results of the study indicated that kigelinone, levofuraltadone, 2-hydroxychrasophanol and isoathyriolin the fungal extract have significant binding modes, with best binding energy scores with their respective antibacterial and anticancer target proteins. Alternaria alternata resident in J. heynei offers a promising source of broad-spectrum antibacterial and anticancer compounds.

Dissemination of antimicrobial resistance and Potassium Tellurite (PT) in the hospital environment: can the use of means with Tellurite and the detection of insertion sequences contribute to prevention and intervention?

Multidrug resistance is a public health problem. Enterobacteriaceae strains may be reservoirs of antimicrobial and potassium tellurite (PT) resistance genes, which is a determinant of bacterial escape to intraphagocytic killing mechanisms. The mobility of resistance genes and their genetic rearrangements are significantly involved in the dissemination of these genes. Mobile genetic elements such as integrons and transposons, as well as IS26 (considered among the predominant ISs), facilitate the formation and mobilization of sets of resistance genes in plasmids capable of transferring these determinants of resistance and virulence horizontally. There is evidence of associations, in plasmids, of genes encoding adhesins, toxins and antimicrobial resistance, possibly related to PT resistance. In the present work, 342 samples were collected for bacteriological culture in different units of a University Hospital in Rio de Janeiro, Brazil (Hospital Kitchen, Medical and Surgical Clinics Wards, and Intensive Care Unit): 98 samples from hands of professionals, 85 hand samples from food handlers, 104 stool samples (rectal swab) from patients and 55 food samples. We isolated 27 strains of Enterobacteriaceae through selective media containing cephalothin (32µg/mL). Bacterial identification was performed by conventional phenotypic methods. Antimicrobial susceptibility tests, as well as the phenotypic test for Extended-Spectrum Beta-lactamases (ESBLs) production, were performed by disk diffusion. PT resistance was also tested by direct seeding in media with 25µg/mL and 112 µg/mL of the compound. Plasmids were visualized by agarose gel electrophoresis of bacterial DNA extracts. Additionally, antimicrobial resistance genes and IS26 were detected by PCR assays. From a total of 27 strains of Enterobacteriaceae isolated, eight strains of Enterobacteriaceae were selected, considering phenotypes indicative of ESBLs production, resistance to aminoglycosides and presence of plasmids of high and low molecular weight (from the hands of food handlers, food and feces from hospitalized patients). Three strains 3/8 (37.5%) were resistant to aminoglycosides and cephalosporins, concomitantly. Four 4/8 strains (50.0%) were positive for the phenotypic test for ESBL production and six 6/8 strains (75.0%) were resistant to PT. Strains with resistance to aminoglycosides and producing ESBL showed amplification product for the genes tested. We consider that these antimicrobial resistance genes, also the determinants for resistance to PT (as a bacterial virulence factor), as well as IS26, may contribute to the plasticity and fitness of R plasmids, in different environments, favoring the circulation of relevant pathogenic strains.

Comparative genomic and phenotypic analyses of the virulence potential in Shiga toxin-producing Escherichia coli O121:H7 and O121:H10.

Shiga toxin-producing Escherichia coli (STEC) O121 is among the top six non-O157 serogroups that are most frequently associated with severe disease in humans. While O121:H19 is predominant, other O121 serotypes have been frequently isolated from environmental samples, but their virulence repertoire is poorly characterized. Here, we sequenced the complete genomes of two animal isolates belonging to O121:H7 and O121:H10 and performed comparative genomic analysis with O121:H19 to assess their virulence potential. Both O121:H7 and O121:H10 strains carry a genome comparable in size with the O121:H19 genomes and belong to phylogroup B1. However, both strains appear to have evolved from a different lineage than the O121:H19 strains according to the core genes-based phylogeny and Multi Locus Sequence Typing. A systematic search of over 300 E. coli virulence genes listed in the Virulence Factor DataBase revealed a total of 73 and 71 in O121:H7 and O121:H10 strains, respectively, in comparison with an average of 135 in the O121:H19 strains. This variation in the virulence genes repertoire was mainly attributed to the reduction in the number of genes related to the Type III Secretion System in the O121:H7 and O121:H10 strains. Compared to the O121:H19 strains, the O121:H7 strain carries more adherence and toxin genes while the O121:H10 strain carries more genes related to the Type VI Secretion System. Although both O121:H7 and O121:H10 strains carry the large virulence plasmid pEHEC, they do not harbor all pEHEC virulence genes in O121:H19. Furthermore, unlike the O121:H19 strains, neither the O121:H7 nor O121:H10 strain carried the Locus of Enterocyte Effacement, OI-122, nor the tellurite resistance island. Although an incomplete Locus of Adhesion and Autoaggregation (LAA) was identified in the O121:H7 and O121:H10 strains, a limited number of virulence genes were present. Consistently, both O121:H7 and O121:H10 strains displayed significant reduced cytotoxicity than either the O157:H7 strain EDL933 or the O121:H19 strain RM8352. In fact, the O121:H7 strain RM8082 appeared to cause minimal cytotoxicity to Vero cells. Our study demonstrated distinct evolutionary lineages among the strains of serotypes O121:H19, O121:H10, and O121:H7 and suggested reduced virulence potentials in STEC strains of O121:H10 and O121:H7.

Identification of hypervirulent Klebsiella pneumoniae carrying terW gene by MacConkey-potassium tellurite medium in the general population.

ObjectivesTo establish a MacConkey-potassium tellurium medium-based method for selectively culturing terW gene-positive Klebsiella pneumoniae (KP), to evaluate its performance and apply it to identifying particular clonal hypervirulent KP (hvKP) strains in epidemiological surveillance.MethodsThe virulence genes, rmpA, iutA, and terW, were detected by PCR. The minimum inhibitory concentration of potassium tellurite of hvKP (rmpA+/ iutA+) and classical KP (rmpA− and iutA−) was determined using the agar dilution method. The MacConkey medium containing 4 μg/ml potassium tellurite was prepared and the performance in detecting terW+ KP was evaluated, including an agreement with PCR and positive/negative predictive value. Fecal samples from healthy volunteers in Fujian were collected and cultured in the medium, then positive strains were identified using MALDI-TOF MS, antimicrobial susceptibility was tested by Kirby-Bauer assays, and virulence genes and capsular serotype genes were tested by PCR.ResultsIn KP isolated from clinical specimens (N = 198), the positive rate of terW was 37.9%, and the detection rate of terW in hvKP was significantly higher than that in classical KP (70.6% vs 13.3%). The potassium tellurite resistance levels of terW+ (N = 75) and terW− (N = 55) KP were 8–128 μg/ml and <1–8 μg/ml, respectively, with significant differences. KP was selectively cultured on a MacConkey medium with 4 μg/ml potassium tellurite, and its agreement with PCR was good (Kappa=0.936), and the positive and negative percent agreement and positive and negative predictive values were 100% (75/75), 92.7% (51/55), 94.9% (75/79), and 100% (51/51), respectively. The prevalence of tellurite-resistant KP was 16.7% (86/516) in fecal samples from healthy volunteers, among which the positive rate of terW was 100% (86/86). The antimicrobial resistance characteristics of terW+ KP showed no difference between healthy volunteers and inpatients. The most common capsular serotypes associated with high virulence were K1, K2, and K57.ConclusionsThe MacConkey medium containing 4 μg/ml potassium tellurite could easily select and culture terW+ KP in fecal samples with high sensitivity and specificity, which is a practical method for the epidemic surveillance of hvKP in the general population.

Potential of tellurite resistance in heterotrophic bacteria from mining environments

SummaryUntreated mining wastes and improper disposal of high-tech devices generate an environmental increase of bioavailable metalloids, exerting stress on autochthonous microbial populations. Tellurium is a metalloid, an element with raising economic importance; nevertheless, its interaction with living organisms is not yet fully understood. Here we characterized aerobic heterotrophic bacteria, isolated from high metal-content mining residues, able to resist/reduce tellurite into tellurium structures and to determine the presence of confirmed tellurite resistance genetic determinants in resistant strains. We identified over 50 tellurite-resistant strains, among 144 isolates, eight strains reduced tellurite to tellurium at different rates, with the concomitant production of tellurium deposits. Most tellurite resistance genes were found in strains from Bacillales, with the prevalence of genes of the ter operon. This work demonstrated that bacterial isolates, from environments with a persistent selective pressure, are potential candidates for uncovering strategies for tellurite resistance and/or production of valuable Te-containing materials.

Comparative Genomics Applied to Systematically Assess Pathogenicity Potential in Shiga Toxin-Producing Escherichia coli O145:H28.

Shiga toxin-producing Escherichia coli (STEC) O145:H28 can cause severe disease in humans and is a predominant serotype in STEC O145 environmental isolates. Here, comparative genomics was applied to a set of clinical and environmental strains to systematically evaluate the pathogenicity potential in environmental strains. While the core genes-based tree separated all O145:H28 strains from the non O145:H28 reference strains, it failed to segregate environmental strains from the clinical. In contrast, the accessory genes-based tree placed all clinical strains in the same clade regardless of their genotypes or serotypes, apart from the environmental strains. Loss-of-function mutations were common in the virulence genes examined, with a high frequency in genes related to adherence, autotransporters, and the type three secretion system. Distinct differences in pathogenicity islands LEE, OI-122, and OI-57, the acid fitness island, and the tellurite resistance island were detected between the O145:H28 and reference strains. A great amount of genetic variation was detected in O145:H28, which was mainly attributed to deletions, insertions, and gene acquisition at several chromosomal “hot spots”. Our study demonstrated a distinct virulence gene repertoire among the STEC O145:H28 strains originating from the same geographical region and revealed unforeseen contributions of loss-of-function mutations to virulence evolution and genetic diversification in STEC.

Complete genome sequencing of transposon-mediated sulfamethoxazole resistance encoded by the Sul1 gene in multidrug-resistant Nocardia farcinica SZ 1509

The objective of this study is to explore the molecular basis of trimethoprim-sulfamethoxazole (SXT) resistance in Nocardia, an SXT-resistant N. farcinica strain, named SZ 1509, by whole-genome sequencing. Antimicrobial susceptibility testing of SZ 1509 was performed by broth microdilution, Etest, and disk diffusion arrays. Genome sequencing and analysis were performed to discover the SXT resistance determinant and its genetic context. Inverse PCR was conducted to confirm the circular form of the composite transposon. PCR for the sul1 gene was performed among SXT-susceptible isolates. SZ 1509 is resistant to many drugs, especially SXT, with a minimum inhibitory concentration (MIC) of up to 32/608 µg/mL (ratio of 1:19 for trimethoprim: sulfamethoxazole). Its assembled genome consists of one chromosome and four plasmids with a total size of 6 613 629 bp and 71.1% of GC content. The plasmid 2 was found to carry one IS6-composite transposon containing IS6100 carrying the sul1 gene, one tellurite resistance gene TerC, and several transcriptional regulators. Inverse PCR analyses showed its circular form. All 10 SXT-susceptible isolates do not contain sul1. In addition, mutations with strong associations to SXT resistance were not conclusive. This is the first study to elucidate the transposon-mediated sulfamethoxazole resistance in N. farcinica. Our results provide insights on acquired drug resistance of N. farcinica and further suggest that the prevalence and correlation of this resistance's determinants in clinical isolates should be continuously monitored to provide effective clinical management of its resultant diseases.

Comparative resistome and virulome analysis of clinical NDM-1–producing carbapenem-resistant Enterobacter cloacae complex

The Enterobacter cloacae complex (ECC) are causatives of hospital-acquired infections. The antimicrobial resistance (AMR) and virulence profiling of ECC promotes our knowledge for their elimination in clinical settings. We assembled the whole genome of four clinical carbapenem-resistant ECCs and characterized their AMR and virulence profiles using whole genome sequencing. The chromosomes length scaled from minimum 3 949 952 bp (for P2) to maximum 4 976 575 bp (for P3). Strains P1 and P2 belonged to sequence type (ST)182. P3 and P4 belonged to ST477 and ST134, respectively. The blaCTX-M-15 gene was detected in P1 plasmid. P1 and P4 harboured the blaTEM-1 and blaOXA-1 genes. blaNDM-1 was found in P1, P3, and P4. No blaOXA-48, blaKPC, blaVIM, or blaIMP were identified. The plasmids were nontransferable and had IncFIB, IncFII, Col, and IncC incompatibility (Inc) groups . Class 1 integron was detected in all strains. Virulence genes related to biofilms, adhesins, siderophores (aerobactin, enterobactin, and salmochelin), intrinsic antimicrobial efflux pumps, secretory systems type I to VI, environmental and antibiotic stress response , outer membrane proteins, and heavy metal (copper, tellurite, arsenic, and zinc) resistance were found. The number of positive virulence factors was higher for P1 compared with other strains. The accumulation of AMR genes in Enterobacter spp. and their high endurance in hostile environments is a serious health problem. More genomic investigations are required to determine their AMR and virulence genetic reservoirs at the global level.

Characterization of the Tellurite-Resistance Properties and Identification of the Core Function Genes for Tellurite Resistance in Pseudomonas citronellolis SJTE-3.

Tellurite is highly toxic to bacteria and commonly used in the clinical screening for pathogens; it is speculated that there is a potential relationship between tellurite resistance and bacterial pathogenicity. Until now, the core function genes of tellurite resistance and their characteristics are still obscure. Pseudomonas citronellolis SJTE-3 was found able to resist high concentrations of tellurite (250 μg/mL) and formed vacuole-like tellurium nanostructures. The terZABCDE gene cluster located in the large plasmid pRBL16 endowed strain SJTE-3 with the tellurite resistance of high levels. Although the terC and terD genes were identified as the core function genes for tellurite reduction and resistance, the inhibition of cell growth was observed when they were used solely. Interestingly, co-expression of the terA gene or terZ gene could relieve the burden caused by the expression of the terCD genes and recover normal cell growth. TerC and TerD proteins commonly shared the conserved sequences and are widely distributed in many pathogenic bacteria, highly associated with the pathogenicity factors.

Disrupting Biofilm Formation and Antibiotic Resistance in Pseudomonas aeruginosa Using Phage-Delivered Sensitivity Cassettes: A Research Protocol

Introduction: Despite antimicrobial resistance topping the list of global health concerns, the development of novel antibiotics has been nearly abandoned due to strict regulations and dwindling economic incentives in the pharmaceutical industry. There is a critical need for alternative strategies to treat multidrug resistant pathogens like Pseudomonas aeruginosa, a pestilent cause of nosocomial infections. Here, we aim to target adaptive resistance in P. aeruginosa biofilms by inducing hypersensitivity to existing antibiotics through phage-delivery of a particular gene cassette. Previous studies have suggested that the rpoS gene is repressed in P. aeruginosa biofilms and that its deletion is correlated with hypervirulence, increased biofilm thickness and antibiotic resistance. Methods: In this protocol, we aim to explore the effect of inducing rpoS overexpression in P. aeruginosa colonies as a potential method to disrupt biofilm structure and increase sensitivity to tobramycin. Phagemids containing rpoS, an accompanying promoter, and a tellurite resistance gene are delivered by P1 bacteriophages to the biofilm to be shared through horizontal gene transfer (HGT). Tellurite is then administered to induce selective pressure for HGT, by favouring uptake of the phagemids due to the presence of the tellurite resistance gene. Consequently, we can assess the effect of rpoS overexpression on biofilm organization and tobramycin sensitivity using measures from confocal laser scanning microscopy (CLSM). Anticipated Results: Given the hypervirulent effects of rpoS deletion, we expect that forcing rpoS overexpression in P. aeruginosa would result in decreased biofilm thickness compared to controls. Furthermore, the colonies are also expected to have lower cell viability following tobramycin administration. Discussion: Overall, our experiment characterizes the effects of rpoS overexpression on biofilm thickness, cell viability and tobramycin resistance. As such, this protocol may have practical implications for re-sensitization of P.aeroginosa to antibiotics. Conclusion: This would demonstrate a potential for phage-mediated hypersensitization of P. aeruginosa that is adaptable to more practical settings, such as in situ on hospital surfaces.