Harnessing a high-quality Salmonella genome catalog to combat antimicrobial resistance and inform outbreak responses

A cDNA-SSH library was established by suppression subtractive hybridization(SSH) using leaves of Yunnan Oryza rufipogon Griff. treated by Xanthomonas oryzae pv. oryzae. All the positive clones were sequenced, 494 available expressed sequence tags(ESTs) were obtained by cluster analysis. The results of BlastN showed that 417 ESTs had homologous sequences in GenBank database. The BlastX results indicated that 104 ESTs had higher similarity with the unknown proteins and putative proteins, and 341 ESTs had significant protein homologies while 49 ESTs had no protein homologies. The study suggested that a large group of Xanthomonas oryzae pv. oryzae of stress-induced genes were found in cDNA library, which are involved in many metabolism pathways such as energy metabolism,protein metabolism, nucleic acid metabolism, defense and resistance response, signal transduction, photosynthesis and transmembrane transport. We studied the mRNA expression of several ESTs which may be bacterial blight resistance genes in control and Xanthomonas oryzae pv. oryzae stress of Yunnan Oryza rufipogon Griff. by semi-quantitative RT-PCR. The results showed that the expression of the clone No. OR7, OR68 and OR826 EST were induced by Xanthomonas oryzae pv. oryzae stress, and were up-regulated. EST OR826 that had no protein homology in protein database may belong to a class of new bacterial blight resistance genes. The mRNA expression of OR143 could be detected in the leaf of control and Xanthomonas oryzae pv. oryzae stress. Its expression was gradually strengthened 48 hours after Xanthomonas oryzae pv. oryzae stress. We suggested these genes participated in resistance defense response in Yunnan Oryza rufipogon Griff. directly. This research provided theoretical basis for discovery of new bacterial blight resistance genes, and laid the foundation for further studies of the molecular mechanisms of bacterial blight resistance in Yunnan Oryza rufipogon Griff.

Bacterial resistance against antibiotics often involves multiple mechanisms that are interconnected to ensure robust protection. So far, the knowledge about underlying regulatory features of those resistance networks is sparse, since they can hardly be determined by experimentation alone. Here, we present the first computational approach to elucidate the interplay between multiple resistance modules against a single antibiotic and how regulatory network structure allows the cell to respond to and compensate for perturbations of resistance. Based on the response of Bacillus subtilis toward the cell wall synthesis-inhibiting antibiotic bacitracin, we developed a mathematical model that comprehensively describes the protective effect of two well-studied resistance modules (BceAB and BcrC) on the progression of the lipid II cycle. By integrating experimental measurements of expression levels, the model accurately predicts the efficacy of bacitracin against the B. subtilis wild type as well as mutant strains lacking one or both of the resistance modules. Our study reveals that bacitracin-induced changes in the properties of the lipid II cycle itself control the interplay between the two resistance modules. In particular, variations in the concentrations of UPP, the lipid II cycle intermediate that is targeted by bacitracin, connect the effect of the BceAB transporter and the homeostatic response via BcrC to an overall resistance response. We propose that monitoring changes in pathway properties caused by a stressor allows the cell to fine-tune deployment of multiple resistance systems and may serve as a cost-beneficial strategy to control the overall response toward this stressor.IMPORTANCE Antibiotic resistance poses a major threat to global health, and systematic studies to understand the underlying resistance mechanisms are urgently needed. Although significant progress has been made in deciphering the mechanistic basis of individual resistance determinants, many bacterial species rely on the induction of a whole battery of resistance modules, and the complex regulatory networks controlling these modules in response to antibiotic stress are often poorly understood. In this work we combined experiments and theoretical modeling to decipher the resistance network of Bacillus subtilis against bacitracin, which inhibits cell wall biosynthesis in Gram-positive bacteria. We found a high level of cross-regulation between the two major resistance modules in response to bacitracin stress and quantified their effects on bacterial resistance. To rationalize our experimental data, we expanded a previously established computational model for the lipid II cycle through incorporating the quantitative action of the resistance modules. This led us to a systems-level description of the bacitracin stress response network that captures the complex interplay between resistance modules and the essential lipid II cycle of cell wall biosynthesis and accurately predicts the minimal inhibitory bacitracin concentration in all the studied mutants. With this, our study highlights how bacterial resistance emerges from an interlaced network of redundant homeostasis and stress response modules.

Antimicrobial resistance (AMR) is a major 21st century global health challenge. The WHO African Region Member States committed to develop and implement multisectoral national action plans (NAPs) that address AMR, in line with the Global Action Plan on AMR (GAP). The aim of this paper is to present the progress of AMR response in the WHO African Region based on the annual Tracking AMR Country Self-Assessment Survey (TrACSS), with a focus on human health indicators. This was a secondary data analysis of responses from forty-one countries that participated in the 2021 TrACSS. Of the 41 countries that responded to the 2021 TrACSS, 35(85%) have developed NAPs. Fifteen 15(37%) of countries have functional AMR multisector working groups. 55% (21/41) of countries are collating data nationally on AMR surveillance. Forty nine percent of countries conducted small-scale AMR awareness campaigns and 53% (21/41) covered AMR in some pre- and in-service training for human health workers. While 83% of countries reported having laws and regulations on the prescription and sale of antimicrobials, only 32% (13/41) have national systems for monitoring antimicrobial use. Twenty-three (58%, 23/41) reported having Infection Prevention and Control (IPC) programs at select health facilities. Countries have developed and are implementing AMR NAPs. Gaps still exist across key indicators monitored through TrACSS. Effective AMR response requires established functional multisectoral governance mechanisms in the One Health approach; political commitment, sustainable funding, and clear monitoring and reporting is critical.

Oxidative stress serves as an important host/environmental signal that triggers a wide range of responses in microorganisms. Here, we identified an oxidative stress sensor and response regulator in the important multidrug-resistant nosocomial pathogen Enterococcus faecium belonging to the MarR family and called AsrR (antibiotic and stress response regulator). The AsrR regulator used cysteine oxidation to sense the hydrogen peroxide which results in its dissociation to promoter DNA. Transcriptome analysis showed that the AsrR regulon was composed of 181 genes, including representing functionally diverse groups involved in pathogenesis, antibiotic and antimicrobial peptide resistance, oxidative stress, and adaptive responses. Consistent with the upregulated expression of the pbp5 gene, encoding a low-affinity penicillin-binding protein, the asrR null mutant was found to be more resistant to β-lactam antibiotics. Deletion of asrR markedly decreased the bactericidal activity of ampicillin and vancomycin, which are both commonly used to treat infections due to enterococci, and also led to over-expression of two major adhesins, acm and ecbA, which resulted in enhanced in vitro adhesion to human intestinal cells. Additional pathogenic traits were also reinforced in the asrR null mutant including greater capacity than the parental strain to form biofilm in vitro and greater persistance in Galleria mellonella colonization and mouse systemic infection models. Despite overexpression of oxidative stress-response genes, deletion of asrR was associated with a decreased oxidative stress resistance in vitro, which correlated with a reduced resistance to phagocytic killing by murine macrophages. Interestingly, both strains showed similar amounts of intracellular reactive oxygen species. Finally, we observed a mutator phenotype and enhanced DNA transfer frequencies in the asrR deleted strain. These data indicate that AsrR plays a major role in antimicrobial resistance and adaptation for survival within the host, thereby contributes importantly to the opportunistic traits of E. faecium.

On the resistance to bacterial leaf blight, caused by Xanthomonas campetris pv. oryzae (Ishiyama 1922) Dye 1978 of Japan, the mode of inheritance and allelic relationships of the genes in three resistant IRRI rice verieties IR28, IR 29 and IR30 were studied. Analysis of the F1 and F2 populations from the crosses between the IRRI varities and susceptible Japanese varieties Toyonishiki, Todorokiwase and Kinmaze revealed that in these IRRI varieties two major genes controlled the resistance to the bacterial groups I and V, and these two genes linked very closely with a recombination value of 2.1% Evidences from allelism tests indicated that these genes were located on the identical loci with two known genes of Kogyoku, Xa-1 and Xa-kg respectively. Howvever, the IRRI varieties were different from Kogyoku in the response of resistance at seedling stage. Hence, it was concluded that those genes should be treated in distinction from Xa-1 and Xa-kg. These results suggest that the gene of the IRRI varieties resistant to bacterial group I and the gene resistant to bacterial group V are multiple alleles at Xa-1 and Xa-kg loci, respectively. The former gene was designated as Xa-1h and the latter gene as Xa-kgh. The significance of these newly-discovered genes was discussed from the standpoint of breeding for bacterial leaf blight resistance.

Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumefaciens-mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2-mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Co-immunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Co-immunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of Bs2. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1-containing protein complex that is required for the expression of bacterial disease resistance.

Amphibian skin is a rich source of natural compounds with diverse antimicrobial and immune defense properties. Our previous studies showed that the frog skin secretions obtained by skin micro-organs from various species of Colombian anurans have antimicrobial activities against bacteria and viruses. We purified for the first time two antimicrobial peptides from the skin micro-organs of the Orinoco lime treefrog (Sphaenorhynchus lacteus) that correspond to Buforin II (BF2) and Frenatin 2.3S (F2.3S). Here, we have synthesized the two peptides and tested them against Gram-negative and Gram-positive bacteria, observing an effective bactericidal activity at micromolar concentrations. Evaluation of BF2 and F2.3S membrane destabilization activity on bacterial cell cultures and synthetic lipid bilayers reveals a distinct membrane interaction mechanism. BF2 agglutinates E. coli cells and synthetic vesicles, whereas F2.3S shows a high depolarization and membrane destabilization activities. Interestingly, we found that F2.3S is able to internalize within bacterial cells and can bind nucleic acids, as previously reported for BF2. Moreover, bacterial exposure to both peptides alters the expression profile of genes related to stress and resistance response. Overall, these results show the multifaceted mechanism of action of both antimicrobial peptides that can provide alternative tools in the fight against bacterial resistance.

Group B Streptococcus (GBS; Streptococcus agalactiae) is a leading cause of sepsis in neonates and pregnant mothers worldwide. Whereas the hyper-virulent serogroup III clonal cluster 17 has been associated with neonatal disease and meningitis, serogroup III ST283 was recently implicated in invasive disease among non-pregnant adults in Asia. Here, through comparative genome analyses of invasive and non-invasive ST283 strains, we identified a truncated DNA-binding regulator of a two-component system in a non-invasive strain that was homologous to Bacillus subtilis bceR, encoding the bceRSAB response regulator, which was conserved among GBS strains. Using isogenic knockout and complementation mutants of the ST283 strain, we demonstrated that resistance to bacitracin and the human antimicrobial peptide cathelicidin LL-37 was reduced in the ΔbceR strain with MICs changing from 64 and 256 μg/ml to 0.25 and 64 μg/ml, respectively. Further, the ATP-binding cassette transporter was upregulated by sub-inhibitory concentrations of bacitracin in the wild-type strain. Upregulation of dltA in the wild-type strain was also observed and thought to explain the increased resistance to antimicrobial peptides. DltA, an enzyme involved in D-alanylation during the synthesis of wall teichoic acids, which mediates reduced antimicrobial susceptibility, was previously shown to be regulated by the bceR-type regulator in Staphylococcus aureus. In a murine infection model, we found that the ΔbceR mutation significantly reduced the mortality rate compared to that with the wild-type strain (p < 0.01). Moreover, this mutant was more susceptible to oxidative stress compared to the wild-type strain (p < 0.001) and was associated with reduced biofilm formation (p < 0.0001). Based on 2-DGE and mass spectrometry, we showed that downregulation of alkyl hydroperoxide reductase (AhpC), a Gls24 family stress protein, and alcohol dehydrogenase (Adh) in the ΔbceR strain might explain the attenuated virulence and compromised stress response. Together, we showed for the first time that the bceR regulator in GBS plays an important role in bacitracin and antimicrobial peptide resistance, virulence, survival under oxidative stress, and biofilm formation.

The expression and binding properties of the rice WRKY68 protein in the Xa21-mediated resistance response to Xanthomonas oryzae pv. Oryzae

Bacterial blight resistance gene B5 has received little attention since it was first described in 1950. A near-isogenic line (NIL) of Gossypium hirsutum cotton, AcB5, was generated in an otherwise bacterial-blight-susceptible 'Acala 44' background. The introgressed locus B5 in AcB5 conferred strong and broad-spectrum resistance to bacterial blight. Segregation patterns of test crosses under Oklahoma field conditions indicated that AcB5 is likely homozygous for resistance at two loci with partial dominance gene action. In controlled-environment conditions, two of the four copies of B5 were required for effective resistance. Contrary to expectations of gene-for-gene theory, AcB5 conferred high resistance toward isogenic strains of Xanthomonas citri subsp. malvacearum carrying cloned avirulence genes avrB4, avrb7, avrBIn, avrB101, and avrB102, respectively, and weaker resistance toward the strain carrying cloned avrb6. The hypothesis that each B gene, in the absence of a polygenic complex, triggers sesquiterpenoid phytoalexin production was tested by measurement of cadalene and lacinilene phytoalexins during resistant responses in five NILs carrying different B genes, four other lines carrying multiple resistance genes, as well as susceptible Ac44E. Phytoalexin production was an obvious, but variable, response in all nine resistant lines. AcB5 accumulated an order of magnitude more of all four phytoalexins than any of the other resistant NILs. Its total levels were comparable to those detected in OK1.2, a highly resistant line that possesses several B genes in a polygenic background.

NBS-LRR genes constitute one of the largest resistance gene families in plants, which play key roles in resistance to pathogens. Although the identification and characterization of the NBS-LRR gene family has been extensively reported in various species, a comprehensive analysis in eggplant has not been previously documented. In this study, a total of 269 SmNBS genes were identified in the eggplant genome. Based on domain classification and phylogenetic analysis, SmNBSs were divided into three subgroups 231 CNLs (CC-NBS-LRR), 36 TNLs (TIR-NBS-LRR), and 2 RNLs (RPW8-NBS-LRR). Chromosomal mapping analysis revealed an uneven distribution of SmNBSs in clusters across chromosomes, with a predominant presence on chromosomes 10, 11, and 12. Structural analysis identified eight conserved motifs previously reported in SmNBSs, exhibiting high conservation in both amino acid sequences and their order. Evolutionary analysis demonstrated that tandem duplication events mainly contributed to the expansion of SmNBS. Subsequently, qRT-PCR analysis demonstrated that nine SmNBSs exhibited differential expression patterns in response to R. solanacearum stress, with EGP05874.1 potentially involved in the resistance response. In conclusion, this study provides a comprehensive insight into SmNBSs, which will enhance the research on eggplant disease resistance and facilitate the breeding of new disease-resistant varieties.

JASMONATE ZIM-domain (JAZ) proteins act as transcriptional repressors of jasmonic acid (JA) responses and play a crucial role in the regulation of host immunity in plants. Here, we report that OsMYC2, a JAZ-interacting transcription factor in rice (Oryza sativa L.), plays an important role in the resistance response against rice bacterial blight, which is one of the most serious diseases in rice, caused by Xanthomonas oryzae pv. oryzae (Xoo). The results showed that OsMYC2 interacted with some OsJAZ proteins in a JAZ-interacting domain (JID)-dependent manner. The up-regulation of OsMYC2 in response to JA was regulated by OsJAZ8. Transgenic rice plants overexpressing OsMYC2 exhibited a JA-hypersensitive phenotype and were more resistant to Xoo. A large-scale microarray analysis revealed that OsMYC2 up-regulated OsJAZ10 as well as many other defense-related genes. OsMYC2 selectively bound to the G-box-like motif of the OsJAZ10 promoter in vivo and regulated the expression of early JA-responsive genes, but not of late JA-responsive genes. The nuclear localization of OsMYC2 depended on a nuclear localization signal within JID. Overall, we conclude that OsMYC2 acts as a positive regulator of early JA signals in the JA-induced resistance against Xoo in rice.

Listeriosis is a severe zoonotic disease caused by Listeria monocytogenes (L. monocytogenes), which can be acquired through animal source foods. This pathogen shows unique virulence fitness allowing it to penetrate and survive inside host cells causing extremely dangerous symptoms. Therefore, we aimed to correlate the clinical outcomes with the antimicrobial resistance and virulence profiles of L. monocytogenes. This is crucial for improving disease management, developing new therapeutic strategies, enhancing public health and food safety, and advancing scientific knowledge. Therefore, we assessed the antimicrobial resistance and virulence profiles of L. monocytogenes isolated from various sources, along with their potential to cause disease, using an in vivo rabbit model. Based on identification criteria, 47 L. monocytogenes isolates (15.7%) were recovered with the highest detection rates among rabbits (22%). Unfortunately, all the investigated isolates showed multidrug resistance (MDR) as well as multi-virulent profiles with 45 highly heterogeneous clusters. Noteworthy, the degree of illnesses of the experimentally infected rabbits was dependent on the virulence profiles. Specific nervous manifestations and severe histopathological alterations were observed in experimental rabbits infected with highly virulent isolates confirming that the potential ability of this pathogen to produce a disease is not always decipherable from its virulence arrays. Finally, it was confirmed that managing infections caused by L. monocytogenes has become increasingly challenging due to its high antimicrobial resistance, strong virulence, and the severe pathological effects linked to its virulence factors.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-10093-z.

Response of microbial antibiotic resistance to pesticides: An emerging health threat

Background Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Many patients experience multiple episodes of GNB-BSI for unclear reasons. This study examines the genetic differences between initial and subsequent isolates of recurrent GNB-BSI. Methods We used a prospective cohort of patients with GNB-BSI at Duke Hospital to identify patients with &amp;gt;1 episode of GNB-BSI due to the same bacterial species. We performed pulsed field gel electrophoresis (PFGE) on paired isolates to determine if the events were Reinfection (paired isolates different) or Relapse (paired isolates genetically identical). We then used whole genome sequencing (WGS) to verify the PFGE findings and explore the genetic similarity between initial and relapsed GNB-BSI isolates. Results Among 1,423 unique patients with GNB-BSI, 60 (4%) experienced recurrent GNB-BSI with the same bacterial species. We performed genotyping (PFGE, followed by WGS) on the paired bacterial isolates from the index and recurrent bloodstream infections for the four most common species in our study population (Escherichia coli, Klebsiella species, Pseudomonas aeruginosa, and Serratia marcescens) (n=48 pairs). We determined that 63% (30/48) of recurrent GNB-BSI episodes were due to relapse and 37% (18/48) were due to reinfection. PFGE correctly identified relapse versus reinfection in 98% (47/48) of cases. The WGS data illustrated the microevolution of relapsed GNB-BSI isolates within patients. When comparing initial to relapsed isolates, we calculated a median of 5 single-nucleotide polymorphisms (SNPs) in relapsed E. coli isolates and 2 SNPs in Klebsiella spp. isolates. Of these SNPs, 37% (E. coli) and 12% (Klebsiella) were non-synonymous changes in coding regions, most commonly genes associated with antimicrobial resistance (AMR), energy metabolism and stress response. Alterations in the AMR profile was common, with 20/30 relapsed isolates demonstrating either loss or acquisition of AMR. Figure 1:Time from initial to recurrent episode of gram-negative bloodstream infection. Relapse (i.e., same bacterial strain noted by green circle) versus reinfection (i.e., different bacterial strain noted by red circle) was determined by pulse field gel electrophoresis followed by WGS. Whole genome sequencing was performed on the initial and recurrent isolate from patients with relapsed GNB-BSI. We identified single nucleotide polymorphisms (SNPs) present in the recurrent isolate, which were not present in the initial isolate. We identified the function of genes containing SNPs in E. coli (n=28 SNPs) and Klebsiella spp. (n=13 SNPs). Synonymous mutations, insertions, deletions, and non-synonymous mutations in hypothetical proteins were excluded. Conclusion Most recurrent GNB-BSI with same species are due to relapse. PFGE has comparable accuracy to WGS to determine reinfection versus relapse. Multiple mutations in genes involved in stress response, AMR and metabolism were identified in relapsed isolates. Disclosures Vance G. Fowler, Jr, MD, MHS, Affinergy: Grant/Research Support|Affinergy: Honoraria|Affinium: Honoraria|Amphliphi Biosciences: Honoraria|ArcBio: Stocks/Bonds|Basilea: Grant/Research Support|Basilea: Honoraria|Bayer: Honoraria|C3J: Honoraria|Cerexa/Forest/Actavis/Allergan: Grant/Research Support|Contrafect: Grant/Research Support|Contrafect: Honoraria|Cubist/Merck: Grant/Research Support|Debiopharm: Grant/Research Support|Deep Blue: Grant/Research Support|Destiny: Honoraria|Genentech: Grant/Research Support|Genentech: Honoraria|Integrated Biotherapeutics: Honoraria|Janssen: Grant/Research Support|Janssen: Honoraria|Karius: Grant/Research Support|Medicines Co.: Honoraria|MedImmune: Grant/Research Support|MedImmune: Honoraria|NIH: Grant/Research Support|Novartis: Grant/Research Support|Novartis: Honoraria|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Regeneron: Honoraria|Sepsis diagnostics: Sepsis diagnostics patent pending|UpToDate: Royalties|Valanbio: Stocks/Bonds.