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516900.9096Genetic Adaptation and Acquisition of Macrolide Resistance in Haemophilus spp. during Persistent Respiratory Tract Colonization in Chronic Obstructive Pulmonary Disease (COPD) Patients Receiving Long-Term Azithromycin Treatment. Patients with chronic obstructive pulmonary disease (COPD) benefit from the immunomodulatory effect of azithromycin, but long-term administration may alter colonizing bacteria. Our goal was to identify changes in Haemophilus influenzae and Haemophilus parainfluenzae during azithromycin treatment. Fifteen patients were followed while receiving prolonged azithromycin treatment (Hospital Universitari de Bellvitge, Spain). Four patients (P02, P08, P11, and P13) were persistently colonized by H. influenzae for at least 3 months and two (P04 and P11) by H. parainfluenzae. Isolates from these patients (53 H. influenzae and 18 H. parainfluenzae) were included to identify, by whole-genome sequencing, antimicrobial resistance changes and genetic variation accumulated during persistent colonization. All persistent lineages isolated before treatment were azithromycin-susceptible but developed resistance within the first months, apart from those belonging to P02, who discontinued the treatment. H. influenzae isolates from P08-ST107 acquired mutations in 23S rRNA, and those from P11-ST2480 and P13-ST165 had changes in L4 and L22. In H. parainfluenzae, P04 persistent isolates acquired changes in rlmC, and P11 carried genes encoding MefE/MsrD efflux pumps in an integrative conjugative element, which was also identified in H. influenzae P11-ST147. Other genetic variation occurred in genes associated with cell wall and inorganic ion metabolism. Persistent H. influenzae strains all showed changes in licA and hgpB genes. Other genes (lex1, lic3A, hgpC, and fadL) had variation in multiple lineages. Furthermore, persistent strains showed loss, acquisition, or genetic changes in prophage-associated regions. Long-term azithromycin therapy results in macrolide resistance, as well as genetic changes that likely favor bacterial adaptation during persistent respiratory colonization. IMPORTANCE The immunomodulatory properties of azithromycin reduce the frequency of exacerbations and improve the quality of life of COPD patients. However, long-term administration may alter the respiratory microbiota, such as Haemophilus influenzae, an opportunistic respiratory colonizing bacteria that play an important role in exacerbations. This study contributes to a better understanding of COPD progression by characterizing the clinical evolution of H. influenzae in a cohort of patients with prolonged azithromycin treatment. The emergence of macrolide resistance during the first months, combined with the role of Haemophilus parainfluenzae as a reservoir and source of resistance dissemination, is a cause for concern that may lead to therapeutic failure. Furthermore, genetic variations in cell wall and inorganic ion metabolism coding genes likely favor bacterial adaptation to host selective pressures. Therefore, the bacterial pathoadaptive evolution in these severe COPD patients raise our awareness of the possible spread of macrolide resistance and selection of host-adapted clones.202336475849
843810.9006Virulence of Bacteria Colonizing Vascular Bundles in Ischemic Lower Limbs. BACKGROUND: We documented previously the presence of bacterial flora in vascular bundles, lymphatics, and lymph nodes of ischemic lower limbs amputated because of multifocal atheromatic changes that made them unsuitable for reconstructive surgery and discussed their potential role in tissue destruction. The question arose why bacterial strains inhabiting lower limb skin and considered to be saprophytes become pathogenic once they colonize deep tissues. Bacterial pathogenicity is evoked by activation of multiple virulence factors encoded by groups of genes. METHODS: We identified virulence genes in bacteria cultured from deep tissue of ischemic legs of 50 patients using a polymerase chain reaction technique. RESULTS: The staphylococcal virulence genes fnbA (fibronectin-binding protein A), cna (collagen adhesin precursor), and ica (intercellular adhesion) were present in bacteria isolated from both arteries and, to a lesser extent, skin. The IS256 gene, whose product is responsible for biofilm formation, was more frequent in bacteria retrieved from the arteries than skin bacteria. Among the virulence genes of Staphylococcus epidermidis encoding autolysin atlE, icaAB (intercellular adhesion), and biofilm insert IS256, only the latter was detected in arterial specimens. Bacteria cultured from the lymphatics did not reveal expression of eta and IS256 in arteries. The Enterococcus faecalis asa 373 (aggregation substance) and cylA (cytolysin activator) frequency was greater in arteries than in skin bacteria, as were the E. faecium cyl A genes. All Pseudomonas aeruginosa virulence genes were present in bacteria cultured from both the skin and arteries. Staphylococci colonizing arterial bundles and transported to tissues via ischemic limb lymphatics expressed virulence genes at greater frequency than did those dwelling on the skin surface. Moreover, enterococci and Pseudomonas isolated from arterial bundles expressed many virulence genes. CONCLUSIONS: These findings may add to the understanding of the mechanism of development of destructive changes in lower limb ischemic tissues by the patient's, but not hospital-acquired, bacteria, as well as the generally unsatisfactory results of antibiotic administration in these cases. More aggressive antibiotic therapy targeted at the virulent species should be applied.201626431369
82820.8995Screening for Resistant Bacteria, Antimicrobial Resistance Genes, Sexually Transmitted Infections and Schistosoma spp. in Tissue Samples from Predominantly Vaginally Delivered Placentae in Ivory Coast and Ghana. Medical complications during pregnancy have been frequently reported from Western Africa with a particular importance of infectious complications. Placental tissue can either become the target of infectious agents itself, such as, e.g., in the case of urogenital schistosomiasis, or be subjected to contamination with colonizing or infection-associated microorganisms of the cervix or the vagina during vaginal delivery. In the retrospective cross-sectional assessment presented here, the quantitative dimension of infection or colonization with selected resistant or pathogenic bacteria and parasites was regionally assessed. To do so, 274 collected placental tissues from Ivory Coastal and Ghanaian women were subjected to selective growth of resistant bacteria, as well as to molecular screening for beta-lactamase genes, Schistosoma spp. and selected bacterial causative agents of sexually transmitted infections (STI). Panton-Valentine-negative methicillin-resistant Staphylococcus aureus (MRSA) was grown from 1.8% of the tissue samples, comprising the spa types t008 and t688, as well as the newly detected ones, t12101 (n = 2) and t12102. While the culture-based recovery of resistant Enterobacterales and nonfermentative rod-shaped Gram-negative bacteria failed, molecular assessments confirmed beta-lactamase genes in 31.0% of the samples with multiple detections of up to four resistance genes per sample and bla(CTX-M), bla(IMP), bla(GES), bla(VIM), bla(OXA-58)-like, bla(NDM), bla(OXA-23)-like, bla(OXA-48)-like and bla(KPC) occurring in descending order of frequency. The beta-lactamase genes bla(OXA-40/24)-like, bla(NMC_A/IMI), bla(BIC), bla(SME), bla(GIM) and bla(DIM) were not detected. DNA of the urogenital schistosomiasis-associated Schistosoma haematobium complex was recorded in 18.6% of the samples, but only a single positive signal for S. mansoni with a high cycle-threshold value in real-time PCR was found. Of note, higher rates of schistosomiasis were observed in Ghana (54.9% vs. 10.3% in Ivory Coast) and Cesarean section was much more frequent in schistosomiasis patients (61.9% vs. 14.8% in women without Schistosoma spp. DNA in the placenta). Nucleic acid sequences of nonlymphogranuloma-venereum-associated Chlamydia trachomatis and of Neisseria gonorrhoeae were recorded in 1.1% and 1.9% of the samples, respectively, while molecular attempts to diagnose Treponema pallidum and Mycoplasma genitalium did not lead to positive results. Molecular detection of Schistosoma spp. or STI-associated pathogens was only exceptionally associated with multiple resistance gene detections in the same sample, suggesting epidemiological distinctness. In conclusion, the assessment confirmed considerable prevalence of urogenital schistosomiasis and resistant bacterial colonization, as well as a regionally expected abundance of STI-associated pathogens. Continuous screening offers seem advisable to minimize the risks for the pregnant women and their newborns.202337623959
517030.8989Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori. BACKGROUND: Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism. RESULTS: The minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4-10 microg/ml was higher than that in strains with the MICs of 1-3 microg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs. CONCLUSION: The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.200919594901
866940.8987The ins and outs of metal homeostasis by the root nodule actinobacterium Frankia. BACKGROUND: Frankia are actinobacteria that form a symbiotic nitrogen-fixing association with actinorhizal plants, and play a significant role in actinorhizal plant colonization of metal contaminated areas. Many Frankia strains are known to be resistant to several toxic metals and metalloids including Pb(2+), Al(+3), SeO2, Cu(2+), AsO4, and Zn(2+). With the availability of eight Frankia genome databases, comparative genomics approaches employing phylogeny, amino acid composition analysis, and synteny were used to identify metal homeostasis mechanisms in eight Frankia strains. Characterized genes from the literature and a meta-analysis of 18 heavy metal gene microarray studies were used for comparison. RESULTS: Unlike most bacteria, Frankia utilize all of the essential trace elements (Ni, Co, Cu, Se, Mo, B, Zn, Fe, and Mn) and have a comparatively high percentage of metalloproteins, particularly in the more metal resistant strains. Cation diffusion facilitators, being one of the few known metal resistance mechanisms found in the Frankia genomes, were strong candidates for general divalent metal resistance in all of the Frankia strains. Gene duplication and amino acid substitutions that enhanced the metal affinity of CopA and CopCD proteins may be responsible for the copper resistance found in some Frankia strains. CopA and a new potential metal transporter, DUF347, may be involved in the particularly high lead tolerance in Frankia. Selenite resistance involved an alternate sulfur importer (CysPUWA) that prevents sulfur starvation, and reductases to produce elemental selenium. The pattern of arsenate, but not arsenite, resistance was achieved by Frankia using the novel arsenite exporter (AqpS) previously identified in the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Based on the presence of multiple tellurite resistance factors, a new metal resistance (tellurite) was identified and confirmed in Frankia. CONCLUSIONS: Each strain had a unique combination of metal import, binding, modification, and export genes that explain differences in patterns of metal resistance between strains. Frankia has achieved similar levels of metal and metalloid resistance as bacteria from highly metal-contaminated sites. From a bioremediation standpoint, it is important to understand mechanisms that allow the endosymbiont to survive and infect actinorhizal plants in metal contaminated soils.201425495525
306950.8985The hospital sink drain biofilm resistome is independent of the corresponding microbiota, the environment and disinfection measures. In hospitals, the transmission of antibiotic-resistant bacteria (ARB) may occur via biofilms present in sink drains, which can lead to infections. Despite the potential role of sink drains in the transmission of ARB in nosocomial infections, routine surveillance of these drains is lacking in most hospitals. As a result, there is currently no comprehensive understanding of the transmission of ARB and the dissemination of antimicrobial resistance genes (ARGs) and associated mobile genetic elements (MGEs) via sink drains. This study employed a multifaceted approach to monitor the total aerobic bacteria as well as the presence of carbapenemase-producing Enterobacterales (CPEs), the microbiota and the resistome of sink drain biofilms (SDBs) and hospital wastewater (WW) of two separate intensive care units (ICUs) in the same healthcare facility in France. Samples of SDB and WW were collected on a monthly basis, from January to April 2023, in the neonatal (NICU) and the adult (AICU) ICUs of Grenoble Alpes University Hospital. In the NICU, sink drain disinfection with surfactants was performed routinely. In the AICU, routine disinfection is not carried out. Culturable aerobic bacteria were quantified on non-selective media, and CPEs were screened using two selective agars. Isolates were identified by MALDI-TOF MS, and antibiotic susceptibility testing (AST) was performed on Enterobacterales and P. aeruginosa. The resistome was analyzed by high-throughput qPCR targeting >80 ARGs and MGEs. The overall bacterial microbiota was assessed via full-length 16S rRNA sequencing. No CPEs were isolated from SDBs in either ICU by bacterial culture. Culture-independent approaches revealed an overall distinct microbiota composition of the SDBs in the two ICUs. The AICU SDBs were dominated by pathogens containing Gram-negative bacterial genera including Pseudomonas, Stenotrophomona, Klebsiella, and Gram-positive Staphylococcus, while the NICU SDBs were dominated by the Gram-negative genera Achromobacter, Serratia, and Acidovorax, as well as the Gram-positive genera Weisella and Lactiplantibacillus. In contrast, the resistome of the SDBs exhibited no significant differences between the two ICUs, indicating that the abundance of ARGs and MGEs is independent of microbiota composition and disinfection practices. The AICU WW exhibited more distinct aerobic bacteria than the NICU WW. In addition, the AICU WW yielded 15 CPEs, whereas the NICU WW yielded a single CPE. All the CPEs were characterized at the species level. The microbiota of the NICU and AICU WW samples differed from their respective SDBs and exhibited distinct variations over the four-month period:the AICU WW contained a greater number of genes conferring resistance to quinolones and integron integrase genes, whereas the NICU WW exhibited a higher abundance of streptogramin resistance genes. Our study demonstrated that the resistome of the hospital SDBs in the two ICUs of the investigated healthcare institute is independent of the microbiota, the environment, and the local disinfection measures. However, the prevalence of CPEs in the WW pipes collecting the waste from the investigated drains differed. These findings offer valuable insights into the resilience of resistance genes in SDBs in ICUs, underscoring the necessity for innovative strategies to combat antimicrobial resistance in clinical environments.202540483807
872360.8984Unraveling the Basis of Neonicotinoid Resistance in Whitefly Species Complex: Role of Endosymbiotic Bacteria and Insecticide Resistance Genes. Bemisia tabaci (whitefly) is one of the most detrimental agricultural insect pests and vectors of many plant viruses distributed worldwide. Knowledge of the distribution patterns and insecticide resistance of this cryptic species is crucial for its management. In this study, genetic variation of mitochondrial cytochrome oxidase subunit 1 (MtCoI) gene of B. tabaci was analyzed followed by a study of the infection profile of various endosymbionts in 26 whitefly populations collected from West Bengal, India. Phylogenetic analysis revealed Asia I as the major cryptic species (65.38%), followed by Asia II 5, China 3, and Asia II 7, which were diversified into 20 different haplotypes. In addition to the primary endosymbiont (C. poriera), each of the four whitefly species showed a variable population of three secondary endosymbionts, majorly Arsenophonus with the highest infection rate (73.07%), followed by Wolbachia and Rickettsia. Further phylogenetic analyses revealed the presence of two subgroups of Arsenophonus, viz., A1 and A2, and one each in Wolbachia (W1) and Rickettsia (R3). Resistance to thiamethoxam, imidacloprid, and acetamiprid insecticides was analyzed for a clear picture of pesticide resistance status. The highest susceptibility was noted toward thiamethoxam (LC(50) = 5.36 mg/L), followed by imidacloprid and acetamiprid. The whitefly population from Purulia and Hooghly districts bearing Asia II 7 and Asia II 5 cryptic species, respectively, shows maximum resistance. The differences in mean relative titer of four symbiotic bacteria among field populations varied considerably; however, a significant positive linear correlation was observed between the resistance level and relative titer of Arsenophonus and Wolbachia in the case of imidacloprid and thiamethoxam, while only Wolbachia was found in case of acetamiprid. Expression analysis demonstrated differential upregulation of insecticide resistance genes with Purulia and Hooghly populations showing maximally upregulated P450 genes. Moreover, thiamethoxam and imidacloprid resistance ratio (RR) showed a significant correlation with CYP6CM1, CYP6DZ7, and CYP4C64 genes, while acetamiprid RR correlated with CYP6CX1, CYP6DW2, CYP6DZ7, and CYP4C64 genes. Taken together, these findings suggested that P450 mono-oxygenase and symbiotic bacteria together affected whitefly resistance to neonicotinoids. Hence, a symbiont-oriented management programme could be a better alternative to control or delay resistance development in whitefly and can be used for pesticide clean-up in an agricultural field.202235814684
843970.8983Comparative genomics analysis and virulence-related factors in novel Aliarcobacter faecis and Aliarcobacter lanthieri species identified as potential opportunistic pathogens. BACKGROUND: Emerging pathogenic bacteria are an increasing threat to public health. Two recently described species of the genus Aliarcobacter, A. faecis and A. lanthieri, isolated from human or livestock feces, are closely related to Aliarcobacter zoonotic pathogens (A. cryaerophilus, A. skirrowii, and A. butzleri). In this study, comparative genomics analysis was carried out to examine the virulence-related, including virulence, antibiotic, and toxin (VAT) factors in the reference strains of A. faecis and A. lanthieri that may enable them to become potentially opportunistic zoonotic pathogens. RESULTS: Our results showed that the genomes of the reference strains of both species have flagella genes (flaA, flaB, flgG, flhA, flhB, fliI, fliP, motA and cheY1) as motility and export apparatus, as well as genes encoding the Twin-arginine translocation (Tat) (tatA, tatB and tatC), type II (pulE and pulF) and III (fliF, fliN and ylqH) secretory pathways, allowing them to secrete proteins into the periplasm and host cells. Invasion and immune evasion genes (ciaB, iamA, mviN, pldA, irgA and fur2) are found in both species, while adherence genes (cadF and cj1349) are only found in A. lanthieri. Acid (clpB), heat (clpA and clpB), osmotic (mviN), and low-iron (irgA and fur2) stress resistance genes were observed in both species, although urease genes were not found in them. In addition, arcB, gyrA and gyrB were found in both species, mutations of which may mediate the resistance to quaternary ammonium compounds (QACs). Furthermore, 11 VAT genes including six virulence (cadF, ciaB, irgA, mviN, pldA, and tlyA), two antibiotic resistance [tet(O) and tet(W)] and three cytolethal distending toxin (cdtA, cdtB, and cdtC) genes were validated with the PCR assays. A. lanthieri tested positive for all 11 VAT genes. By contrast, A. faecis showed positive for ten genes except for cdtB because no PCR assay for this gene was available for this species. CONCLUSIONS: The identification of the virulence, antibiotic-resistance, and toxin genes in the genomes of A. faecis and A. lanthieri reference strains through comparative genomics analysis and PCR assays highlighted the potential zoonotic pathogenicity of these two species. However, it is necessary to extend this study to include more clinical and environmental strains to explore inter-species and strain-level genetic variations in virulence-related genes and assess their potential to be opportunistic pathogens for animals and humans.202235761183
1280.8982A Diketopiperazine, Cyclo-(L-Pro-L-Ile), Derived From Bacillus thuringiensis JCK-1233 Controls Pine Wilt Disease by Elicitation of Moderate Hypersensitive Reaction. Pine wilt disease (PWD) caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus is one of the devastating diseases affecting pine forests worldwide. Although effective control measurements are still missing, induction of resistance could represent a possible eco-friendly alternative. In this study, induced resistance-based in vitro and in vivo screening tests were carried out for selection of bacteria with the ability to suppress PWD. Out of 504 isolated bacteria, Bacillus thuringiensis JCK-1233 was selected for its ability to boost pathogenesis-related 1 (PR1) gene expression, a marker of systemic acquired resistance. Moreover, treatment of pine seedlings with B. thuringiensis JCK-1233 resulted in increased expression of other defense-related genes, and significantly inhibited PWD development under greenhouse conditions. However, B. thuringiensis JCK-1233 showed no direct nematicidal activity against B. xylophilus. To identify the effective compound responsible for the induction of resistance in B. thuringiensis JCK-1233, several diketopiperazines (DPKs) including cyclo-(D-Pro-L-Val), cyclo-(L-Pro-L-Ile), cyclo-(L-Pro-L-Phe), and cyclo-(L-Leu-L-Val) were isolated and tested. Foliar treatment of pine seedlings with Cyclo-(L-Pro-L-Ile) resulted in suppression of PWD severity and increased the expression of defense-related genes similarly to B. thuringiensis JCK-1233 treatment. Interestingly, treatment with B. thuringiensis JCK-1233 or cyclo-(L-Pro-L-Ile) showed moderately enhanced expression of PR-1, PR-2, PR-3, PR-4, PR-5, and PR-9 genes following inoculation with PWN compared to that in the untreated control, indicating that they mitigated the burst of hypersensitive reaction in susceptible pine seedlings. In contrast, they significantly increased the expression levels of PR-6 and PR-10 before PWN inoculation. In conclusion, foliar spraying with either B. thuringiensis JCK-1233 culture suspension or DPKs could induce resistance in pine seedlings, thereby alleviating the serious damage by PWD. Taken together, this study supports aerial spraying with eco-friendly biotic or abiotic agents as a valuable strategy that may mark an epoch for the control of PWD in pine forests.202032849672
80890.8980Exposure of Legionella pneumophila to low-shear modeled microgravity: impact on stress response, membrane lipid composition, pathogenicity to macrophages and interrelated genes expression. Here, we studied the effect of low-shear modeled microgravity (LSMMG) on cross stress resistance (heat, acid, and oxidative), fatty acid content, and pathogenicity along with alteration in expression of stress-/virulence-associated genes in Legionella pneumophila. The stress resistance analysis result indicated that bacteria cultivated under LSMMG environments showed higher resistance with elevated D-values at 55 °C and in 1 mM of hydrogen peroxide (H(2)O(2)) conditions compared to normal gravity (NG)-grown bacteria. On the other hand, there was no significant difference in tolerance (p < 0.05) toward simulated gastric fluid (pH-2.5) acid conditions. In fatty acid analysis, our result showed that a total amount of saturated and cyclic fatty acids was increased in LSMMG-grown cells; as a consequence, they might possess low membrane fluidity. An upregulated expression level was noticed for stress-related genes (hslV, htrA, grpE, groL, htpG, clpB, clpX, dnaJ, dnaK, rpoH, rpoE, rpoS, kaiB, kaiC, lpp1114, ahpC1, ahpC2, ahpD, grlA, and gst) under LSMMG conditions. The reduced virulence (less intracellular bacteria and less % of induce apoptosis in RAW 264.7 macrophages) of L. pneumophila under LSMMG conditions may be because of downregulation related genes (dotA, dotB, dotC, dotD, dotG, dotH, dotL, dotM, dotN, icmK, icmB, icmS, icmT, icmW, ladC, rtxA, letA, rpoN, fleQ, fleR, and fliA). In the LSMMG group, the expression of inflammation-related factors, such as IL-1α, TNF-α, IL-6, and IL-8, was observed to be reduced in infected macrophages. Also, scanning electron microscopy (SEM) analysis showed less number of LSMMG-cultivated bacteria attached to the host macrophages compared to NG. Thus, our study provides understandings about the changes in lipid composition and different genes expression due to LSMMG conditions, which apparently influence the alterations of L. pneumophila' stress/virulence response.202438305908
8472100.8979Genetic architecture of resistance to plant secondary metabolites in Photorhabdus entomopathogenic bacteria. BACKGROUND: Entomopathogenic nematodes of the genus Heterorhabditis establish a symbiotic association with Photorhabdus bacteria. Together, they colonize and rapidly kill insects, making them important biological control agents against agricultural pests. Improving their biocontrol traits by engineering resistance to plant secondary metabolites (benzoxazinoids) in Photorhabdus symbiotic bacteria through experimental evolution has been shown to increase their lethality towards benzoxazinoid-defended larvae of the western corn rootworm, a serious crop pest of maize, and it is therefore a promising approach to develop more efficient biocontrol agents to manage this pest. To enhance our understanding of the genetic bases of benzoxazinoid resistance in Photorhabdus bacteria, we conducted an experimental evolution experiment with a phylogenetically diverse collection of Photorhabdus strains from different geographic origins. We cultured 27 different strains in medium containing 6-methoxy-2-benzoxazolinone (MBOA), a highly active benzoxazinoid breakdown product, for 35 24 h-cycles to select for benzoxazinoid-resistant strains. Then, we carried out genome-wide sequence comparisons to uncover the genetic alterations associated with benzoxazinoid resistance. Lastly, we evaluated the resistance of the newly isolated resistant Photorhabdus strains to eight additional bioactive compounds, including 2-benzoxazolinone (BOA), nicotine, caffeine, 6-chloroacetyl-2-benzoxazolinone (CABOA), digitoxin, fenitrothion, ampicillin, and kanamycin. RESULTS: We found that benzoxazinoid resistance evolves rapidly in Photorhabdus in a strain-specific manner. Across the different Photorhabdus strains, a total of nineteen nonsynonymous point mutations, two stop codon gains, and one frameshift were associated with higher benzoxazinoid resistance. The different genetic alterations were polygenic and occurred in genes coding for the EnvZ/OmpR two-component regulatory system, the different subunits of the DNA-directed RNA polymerase, and the AcrABZ-TolC multidrug efflux pump. Apart from increasing MBOA resistance, the different mutations were also associated with cross-resistance to 2-benzoxazolinone (BOA), nicotine, caffeine, and 6-chloroacetyl-2-benzoxazolinone (CABOA) and with collateral sensitivity to fenitrothion, ampicillin, and kanamycin. Targeted mutagenesis will provide a deeper mechanistic understanding, including the relative contribution of the different mutation types. CONCLUSIONS: Our study reveals several genomic features that are associated with resistance to xenobiotics in this important group of biological control agents and enhances the availability of molecular tools to develop better biological control agents, which is essential for more sustainable and ecologically friendly agricultural practices.202541168779
5143110.8969Genomic Insights Into the Pathogenicity of a Novel Biofilm-Forming Enterococcus sp. Bacteria (Enterococcus lacertideformus) Identified in Reptiles. Whole genome analysis of a novel species of enterococci, Enterococcus lacertideformus, causing multi-systemic and invariably fatal disease in critically endangered Christmas Island reptiles was undertaken to determine the genetic elements and potential mechanisms conferring its pathogenic nature, biofilm-forming capabilities, immune recognition avoidance, and inability to grow in vitro. Comparative genomic analyses with related and clinically significant enterococci were further undertaken to infer the evolutionary history of the bacterium and identify genes both novel and absent. The genome had a G + C content of 35.1%, consisted of a circular chromosome, no plasmids, and was 2,419,934 bp in length (2,321 genes, 47 tRNAs, and 13 rRNAs). Multi-locus sequence typing (MLST), and single nucleotide polymorphism (SNP) analysis of multiple E. lacertideformus samples revealed they were effectively indistinguishable from one another and highly clonal. E. lacertideformus was found to be located within the Enterococcus faecium species clade and was closely related to Enterococcus villorum F1129D based on 16S rDNA and MLST house-keeping gene analysis. Antimicrobial resistance (DfreE, EfrB, tetM, bcrRABD, and sat4) and virulence genes (Fss3 and ClpP), and genes conferring tolerance to metals and biocides (n = 9) were identified. The detection of relatively few genes encoding antimicrobial resistance and virulence indicates that this bacterium may have had no exposure to recently developed and clinically significant antibiotics. Genes potentially imparting beneficial functional properties were identified, including prophages, insertion elements, integrative conjugative elements, and genomic islands. Functional CRISPR-Cas arrays, and a defective prophage region were identified in the genome. The study also revealed many genomic loci unique to E. lacertideformus which contained genes enriched in cell wall/membrane/envelop biogenesis, and carbohydrate metabolism and transport functionality. This finding and the detection of putative enterococcal biofilm determinants (EfaAfs, srtC, and scm) may underpin the novel biofilm phenotype observed for this bacterium. Comparative analysis of E. lacertideformus with phylogenetically related and clinically significant enterococci (E. villorum F1129D, Enterococcus hirae R17, E. faecium AUS0085, and Enterococcus faecalis OG1RF) revealed an absence of genes (n = 54) in E. lacertideformus, that encode metabolic functionality, which potentially hinders nutrient acquisition and/or utilization by the bacterium and precludes growth in vitro. These data provide genetic insights into the previously determined phenotype and pathogenic nature of the bacterium.202133737921
5171120.8968Adaptive laboratory-evolved MRSA with PPEF manifests cross-susceptibility to oxacillin and hypersensitivity to ciprofloxacin. Emerging resistance to current antibiotics is a global threat to human health. Therefore, comprehending the mechanism behind antibiotic resistance holds paramount importance. In the pursuit of finding new antibacterial agents, our group has developed a small molecule, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole), having bisbenzimidazole as a pharmacophore, targeting bacterial type IA topoisomerase, a novel drug target in bacteria. We examined the emergence of mutations leading to PPEF resistance in laboratory-evolved Staphylococcus aureus strains. The growth curve revealed that S. aureus 25923 PPEF-resistant (SA-PR) and methicillin-resistant S. aureus 43300 PPEF-resistant (MRSA-PR) attained stationary phase earlier than their respective reference strains. RNA sequencing analysis revealed that atpD (ATP synthase gene) was downregulated by 2 log(2)-fold in both SA-PR and MRSA-PR strains, whereas there was 10 to 13 log(2)-fold downregulation of mecR1 (methicillin resistance-inducing gene), ble (bleomycin resistance-inducing gene), blaZ (beta-lactamase), pbp (penicillin-binding protein gene), ermA (rRNA adenine methyltransferase gene), and kdpB (potassium-transporting ATPase) in the MRSA-PR strain. Quantitative reverse-transcriptase PCR data confirmed these results. Additionally, MRSA-PR showed a 5 log(2)-fold upregulation of comG and a 9 log(2)-fold downregulation of topB, indicating increased genomic variability and stress adaptation contributing to resistance. Genomic sequencing revealed deletions of resistance genes, including aac(6')-aph(2''), aadD, mecA, and blaZ in MRSA-PR, resulting in a gain in resistance and a diminishing returns epistasis pattern in PPEF-evolved S. aureus strains. This led to the development of an evolved MRSA-PR strain susceptible to oxacillin, ciprofloxacin, gentamicin, and imipenem. Our findings indicate that adaptation to PPEF has increased antibiotic susceptibility, thereby changing the clinical outcomes of infections.IMPORTANCEThis study investigates how Staphylococcus aureus bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) strain, develop resistance to a new candidate antibacterial compound, PPEF (2'-(4-ethoxyphenyl)-5-(4-propylpiperazin-1-yl)-1H,1'H-2,5'-bibenzo(d)imidazole). The research found that resistant strains grew slower and showed significant changes in the activity of genes related to antibiotic resistance. Some resistance genes were deleted in the resistant MRSA strain, making it more sensitive to other antibiotics like oxacillin and ciprofloxacin. These findings highlight how resistance to PPEF leads to increased sensitivity to conventional antibiotics. This suggests that developing combination therapies of PPEF with other antibiotics could optimize treatment regimens and slow resistance evolution. This study also indicates that the antibiotic regimens could be designed to force resistant bacteria into evolutionary trade-offs, where they lose resistance to widely used antibiotics while gaining resistance to a new compound like PPEF.202540662666
1535130.8966Complete Genome Sequencing of Acinetobacter baumannii AC1633 and Acinetobacter nosocomialis AC1530 Unveils a Large Multidrug-Resistant Plasmid Encoding the NDM-1 and OXA-58 Carbapenemases. Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes bla(NDM-1) and bla(OXA-58) in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The bla(NDM-1) gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas bla(OXA-58) was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.202133504662
8445140.8963A genome-wide association study in catfish reveals the presence of functional hubs of related genes within QTLs for columnaris disease resistance. BACKGROUND: Columnaris causes severe mortalities among many different wild and cultured freshwater fish species, but understanding of host resistance is lacking. Catfish, the primary aquaculture species in the United States, serves as a great model for the analysis of host resistance against columnaris disease. Channel catfish in general is highly resistant to the disease while blue catfish is highly susceptible. F2 generation of hybrids can be produced where phenotypes and genotypes are segregating, providing a useful system for QTL analysis. To identify genes associated with columnaris resistance, we performed a genome-wide association study (GWAS) using the catfish 250 K SNP array with 340 backcross progenies derived from crossing female channel catfish (Ictalurus punctatus) with male F1 hybrid catfish (female channel catfish I. punctatus × male blue catfish I. furcatus). RESULTS: A genomic region on linkage group 7 was found to be significantly associated with columnaris resistance. Within this region, five have known functions in immunity, including pik3r3b, cyld-like, adcyap1r1, adcyap1r1-like, and mast2. In addition, 3 additional suggestively associated QTL regions were identified on linkage groups 7, 12, and 14. The resistant genotypes on the QTLs of linkage groups 7 and 12 were found to be homozygous with both alleles being derived from channel catfish. The paralogs of the candidate genes in the suggestively associated QTL of linkage group 12 were found on the QTLs of linkage group 7. Many candidate genes on the four associated regions are involved in PI3K pathway that is known to be required by many bacteria for efficient entry into the host. CONCLUSION: The GWAS revealed four QTLs associated with columnaris resistance in catfish. Strikingly, the candidate genes may be arranged as functional hubs; the candidate genes within the associated QTLs on linkage groups 7 and 12 are not only co-localized, but also functionally related, with many of them being involved in the PI3K signal transduction pathway, suggesting its importance for columnaris resistance.201525888203
5235150.8963Draft genome sequences of rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and MEZEK194 carrying mobile colistin resistance gene mcr-9 isolated from wastewater in South Africa. OBJECTIVES: Antimicrobial-resistant bacteria of the order Enterobacterales are emerging threats to global public and animal health, leading to morbidity and mortality. The emergence of antimicrobial-resistant, livestock-associated pathogens is a great public health concern. The genera Enterobacter and Lelliottia are ubiquitous, facultatively anaerobic, motile, non-spore-forming, rod-shaped Gram-negative bacteria belonging to the Enterobacteriaceae family and include pathogens of public health importance. Here, we report the first draft genome sequences of a rare Lelliottia nimipressuralis strain MEZLN61 and two Enterobacter kobei strains MEZEK193 and MEZEK194 in Africa. METHODS: The bacteria were isolated from environmental wastewater samples. Bacteria were cultured on nutrient agar, and the pure cultures were subjected to whole-genome sequencing. Genomic DNA was sequenced using an Illumina MiSeq platform. Generated reads were trimmed and subjected to de novo assembly. The assembled contigs were analysed for virulence genes, antimicrobial resistance genes, and extra-chromosomal plasmids, and multilocus sequence typing was performed. To compare the sequenced strains with other, previously sequenced E. kobei and L. nimipressuralis strains, available raw read sequences were downloaded, and all sequence files were treated identically to generate core genome bootstrapped maximum likelihood phylogenetic trees. RESULTS: Whole-genome sequencing analyses identified strain MEZLN61 as L. nimipressuralis and strains MEZEK193 and MEZEK194 as E. kobei. MEZEK193 and MEZEK194 carried genes encoding resistance to fosfomycin (fosA), beta-lactam antibiotics (bla(ACT-9)), and colistin (mcr-9). Additionally, MEZEK193 harboured nine different virulence genes, while MEZEK194 harboured eleven different virulence genes. The phenotypic analysis showed that L. nimipressuralis strain MEZLN61 was susceptible to colistin (2 μg/mL), while E. kobei MEZEK193 (64 μg/mL) and MEZEK194 (32 μg/mL) were resistant to colistin. CONCLUSION: The genome sequences of strains L. nimipressuralis MEZLN6, E. kobei MEZEK193, and E. kobei MEZEK194 will serve as a reference point for molecular epidemiological studies of L. nimipressuralis and E. kobei in Africa. In addition, this study provides an in-depth analysis of the genomic structure and offers important information that helps clarify the pathogenesis and antimicrobial resistance of L. nimipressuralis and E. kobei. The detection of mcr-9, which is associated with very low-level colistin resistance in Enterobacter species, is alarming and may indicate the undetected dissemination of mcr genes in bacteria of the order Enterobacterales. Continuous monitoring and surveillance of the prevalence of mcr genes and their associated phenotypic changes in clinically important pathogens and environmentally associated bacteria is necessary to control and prevent the spread of colistin resistance.202336948496
827160.8962Characterization of a ST137 multidrug-resistant Campylobacter jejuni strain with a tet(O)-positive genomic island from a bloodstream infection patient. Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and rarely cause bloodstream infection. Herein, we characterized a multidrug-resistant C. jejuni strain LZCJ isolated from a tumor patient with bloodstream infection. LZCJ was resistant to norfloxacin, ampicillin, ceftriaxone, ciprofloxacin and tetracycline. It showed high survival rate in serum and acidic environment. Whole genome sequencing (WGS) analysis revealed that strain LZCJ had a single chromosome of 1,629,078 bp (30.6 % G + C content) and belonged to the ST137 lineage. LZCJ shared the highest identity of 99.66 % with the chicken-derived C. jejuni MTVDSCj20. Four antimicrobial resistance genes (ARGs) were detected, bla(OXA-61), tet(O), gyrA (T86I), and cmeR (G144D and S207G). In addition, a 12,746 bp genomic island GI_LZCJ carrying 15 open reading frames (ORFs) including the resistance gene tet(O) was identified. Sequence analysis found that the GI_LZCJ was highly similar to the duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. 137 non-synonymous mutations in motility related genes (flgF, fapR, flgS), capsular polysaccharide (CPS) coding genes (kpsE, kpsF, kpsM, kpsT), metabolism associated genes (nuoF, nuoG, epsJ, holB), and transporter related genes (comEA, gene0911) were confirmed in LZCJ compared with the best closed chicken-derived strain MTVDSCj20. Our study showed that C. jejuni strain LZCJ was highly similar to the chicken-derived strain MTVDSCj20 but with a lot of SNPs involved in motility, CPS and metabolism coding genes. This strain possessed a tet(O)-positive genomic island GI_LZCJ, which was closed to duck-derived C. jejuni ZS004, but with an additional ISChh1-like sequence. The above data indicated that the LZCJ strain may originate from foodborne bacteria on animals and the importance of continuous surveillance for the spread of foodborne bacteria.202439208964
5200170.8962Whole genome sequencing of the multidrug-resistant Chryseobacterium indologenes isolated from a patient in Brazil. Chryseobacterium indologenes is a non-glucose-fermenting Gram-negative bacillus. This emerging multidrug resistant opportunistic nosocomial pathogen can cause severe infections in neonates and immunocompromised patients. This study aimed to present the first detailed draft genome sequence of a multidrug-resistant C. indologenes strain isolated from the cerebrospinal fluid of an infant hospitalized at the Neonatal Intensive Care Unit of Brazilian Tertiary Hospital. We first analyzed the susceptibility of C. indologenes strain to different antibiotics using the VITEK 2 system. The strain demonstrated an outstanding resistance to all the antibiotic classes tested, including β-lactams, aminoglycosides, glycylcycline, and polymyxin. Next, C. indologenes was whole-genome-sequenced, annotated using Prokka and Rapid Annotation using Subsystems Technology (RAST), and screened for orthologous groups (EggNOG), gene ontology (GO), resistance genes, virulence genes, and mobile genetic elements using different software tools. The draft genome contained one circular chromosome of 4,836,765 bp with 37.32% GC content. The genomic features of the chromosome present numerous genes related to cellular processes that are essential to bacteria. The MDR C. indologenes revealed the presence of genes that corresponded to the resistance phenotypes, including genes to β-lactamases (bla (IND-13), bla (CIA-3), bla (TEM-116), bla (OXA-209), bla (VEB-15)), quinolone (mcbG), tigecycline (tet(X6)), and genes encoding efflux pumps which confer resistance to aminoglycosides (RanA/RanB), and colistin (HlyD/TolC). Amino acid substitutions related to quinolone resistance were observed in GyrA (S83Y) and GyrB (L425I and K473R). A mutation that may play a role in the development of colistin resistance was detected in lpxA (G68D). Chryseobacterium indologenes isolate harbored 19 virulence factors, most of which were involved in infection pathways. We identified 13 Genomic Islands (GIs) and some elements associated with one integrative and conjugative element (ICEs). Other elements linked to mobile genetic elements (MGEs), such as insertion sequence (ISEIsp1), transposon (Tn5393), and integron (In31), were also present in the C. indologenes genome. Although plasmids were not detected, a ColRNAI replicon type and the most resistance genes detected in singletons were identified in unaligned scaffolds. We provided a wide range of information toward the understanding of the genomic diversity of C. indologenes, which can contribute to controlling the evolution and dissemination of this pathogen in healthcare settings.202235966843
2482180.8962Prophages encoding human immune evasion cluster genes are enriched in Staphylococcus aureus isolated from chronic rhinosinusitis patients with nasal polyps. Prophages affect bacterial fitness on multiple levels. These include bacterial infectivity, toxin secretion, virulence regulation, surface modification, immune stimulation and evasion and microbiome competition. Lysogenic conversion arms bacteria with novel accessory functions thereby increasing bacterial fitness, host adaptation and persistence, and antibiotic resistance. These properties allow the bacteria to occupy a niche long term and can contribute to chronic infections and inflammation such as chronic rhinosinusitis (CRS). In this study, we aimed to identify and characterize prophages present in Staphylococcus aureus from patients suffering from CRS in relation to CRS disease phenotype and severity. Prophage regions were identified using PHASTER. Various in silico tools like ResFinder and VF Analyzer were used to detect virulence genes and antibiotic resistance genes respectively. Progressive MAUVE and maximum likelihood were used for multiple sequence alignment and phylogenetics of prophages respectively. Disease severity of CRS patients was measured using computed tomography Lund-Mackay scores. Fifty-eight S. aureus clinical isolates (CIs) were obtained from 28 CRS patients without nasal polyp (CRSsNP) and 30 CRS patients with nasal polyp (CRSwNP). All CIs carried at least one prophage (average=3.6) and prophages contributed up to 7.7 % of the bacterial genome. Phage integrase genes were found in 55/58 (~95 %) S. aureus strains and 97/211 (~46 %) prophages. Prophages belonging to Sa3int integrase group (phiNM3, JS01, phiN315) (39/97, 40%) and Sa2int (phi2958PVL) (14/97, 14%) were the most prevalent prophages and harboured multiple virulence genes such as sak, scn, chp, lukE/D, sea. Intact prophages were more frequently identified in CRSwNP than in CRSsNP (P=0.0021). Intact prophages belonging to the Sa3int group were more frequent in CRSwNP than in CRSsNP (P=0.0008) and intact phiNM3 were exclusively found in CRSwNP patients (P=0.007). Our results expand the knowledge of prophages in S. aureus isolated from CRS patients and their possible role in disease development. These findings provide a platform for future investigations into potential tripartite associations between bacteria-prophage-human immune system, S. aureus evolution and CRS disease pathophysiology.202134907894
9054190.8959Clinically Relevant Concentrations of Polymyxin B and Meropenem Synergistically Kill Multidrug-Resistant Pseudomonas aeruginosa and Minimize Biofilm Formation. The emergence of antibiotic resistance has severely impaired the treatment of chronic respiratory infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa. Since the reintroduction of polymyxins as a last-line therapy against MDR Gram-negative bacteria, resistance to its monotherapy and recurrent infections continue to be reported and synergistic antibiotic combinations have been investigated. In this study, comprehensive in vitro microbiological evaluations including synergy panel screening, population analysis profiling, time-kill kinetics, anti-biofilm formation and membrane damage analysis studies were conducted to evaluate the combination of polymyxin B and meropenem against biofilm-producing, polymyxin-resistant MDR P. aeruginosa. Two phylogenetically unrelated MDR P. aeruginosa strains, FADDI-PA060 (MIC of polymyxin B [MIC(polymyxin B)], 64 mg/L; MIC(meropenem), 64 mg/L) and FADDI-PA107 (MIC(polymyxin B), 32 mg/L; MIC(meropenem), 4 mg/L) were investigated. Genome sequencing identified 57 (FADDI-PA060) and 50 (FADDI-PA107) genes predicted to confer resistance to a variety of antimicrobials, as well as multiple virulence factors in each strain. The presence of resistance genes to a particular antibiotic class generally aligned with MIC results. For both strains, all monotherapies of polymyxin B failed with substantial regrowth and biofilm formation. The combination of polymyxin B (16 mg/L)/meropenem (16 mg/L) was most effective, enhancing initial bacterial killing of FADDI-PA060 by ~3 log(10) CFU/mL, followed by a prolonged inhibition of regrowth for up to 24 h with a significant reduction in biofilm formation (* p < 0.05). Membrane integrity studies revealed a substantial increase in membrane depolarization and membrane permeability in the surviving cells. Against FADDI-PA107, planktonic and biofilm bacteria were completely eradicated. In summary, the combination of polymyxin B and meropenem demonstrated synergistic bacterial killing while reinstating the efficacy of two previously ineffective antibiotics against difficult-to-treat polymyxin-resistant MDR P. aeruginosa.202133918040