Antibiotic Production Research Articles

Variation in partial direct costs of dry cow therapy on 37 large dairy herds

The objective of this observational study was to estimate partial direct costs of dry cow antibiotic therapy (DCT) protocols used on 37 large dairy herds in Wisconsin and to estimate the potential monetary savings and reduction in antimicrobial usage (AMU) if selective DCT was used. Partial direct costs of DCT were calculated using costs of intramammary (IMM) antimicrobials and teat sealants. Data were retrospectively collected on 37 large dairy farms for a period of one year and included the total number of cows dried-off, types of IMM antimicrobial used, and product prices (obtained from farm invoices). A single farm visit was performed to verify data. Clinical mastitis (CM) and SCC history across lactation were used as the criteria to identify cows eligible to receive only teat sealants (no antibiotic DCT) based on adoption of a hypothetical selective DCT program. Descriptive statistics were performed using PROC MEANS to summarize continuous herd and cow characteristics. Differences in costs among IMM antibiotic products and eligibility to not receive antibiotics at dry off by parity were analyzed using ANOVA. Differences in milk yield at the last test day and DIM at dry-off based on eligibility for selective DCT were analyzed using PROC MIXED. A total of 35,691 cows were dried-off across all herds (n = 37) and most of the herds used IMM antibiotic DCT at dry-off in most of the cows. Teat sealant was used as part of the dry-off protocol in all but 3 herds. Of the enrolled farms, 30 used blanket antibiotic DCT in all quarters of all cows and 7 herds used selective DCT with no administration of antibiotics given to 0.8% to 58% of the dry cows within herd. Across all farms, the average cost of products given per dried cow was $19.57 ± 0.64 and ranged from $8.72 to $24.04. As expected, the cost per dried cow was greater ($23.45 ± 0.38) in herds that used higher-cost intramammary antibiotics as compared with herds that used lower-cost intramammary antibiotics ($16.64 ± 0.40). When using an algorithm based on udder health records to hypothetically select cows that would be eligible to receive teat sealants only (no antibiotic DCT), eligibility ranged from 27.3% to 93.3% within-herds and varied by parity, milk yield at last test day, and DIM at dry-off. If a selective DCT program based on udder health records was used, an overall reduction of $5.37 (27% of total costs) per dry cow would be expected. Likewise, adoption of selective DCT based on udder health records would reduce AMU at dry off by approximately 51%. However, neither of these estimates included potential adverse health complications (such as increased clinical mastitis) that can occur if selective DCT programs are not effectively performed. Variations in partial direct costs at dry-off were observed among herds based on treatment protocols. In herds that have good udder health management and contagious mastitis pathogens controlled, adoption of selective DCT based on SCC and history of clinical mastitis can reduce AMU and partial direct costs of dry cow management.

An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein–protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor.Key points• GarR/GarS is a TCS with domains for signal transduction and response regulation• GarR/GarS is an essential negative regulator of the ACT and RED production• GarR/GarS putatively interacts with and regulates activators of ACT and RED

Identifying and characterizing antibiotic producers found in and on the haustorium of Epifagus virginiana.

Epifagus virginiana, a holoparsite of Fagus grandifolia, utilizes haustoria to parasitize its host. Little is known about the microbiome of this plant's parasitic organ. To shed light on these unexplored ecological interactions we collected eight individual E. virginiana specimens from Kanawha State Forest (Charleston, West Virginia) and used culture-based methods and microscopy to assess total counts and morphotypes, revealing a diverse microbial community on the haustorium surface, including 3 bacterial phyla and fungi. The internal haustorium environment was lower in counts and diversity, indicating the haustorium's internal microbiome is likely a subset of the soil microbiome. We explored the capabilities of these microorganisms to produce bioactive compounds. Fifty-eight morphospecies were investigated for cellulose degradation and antibiotic production. Forty-six isolates were challenged in a cross-streak assay against a Micrococcus roseus (Gram-positive) and Escherichia coli K12(Gram-negative) and 35% showed antagonism for at least one type. Seven surface isolates showed a range of antagonism against M. roseus from 0.85 cm ±0.21 cm to 2.25 cm ±0.07 cm, and 2 against E. coli from 0.75 cm ±0.35 cm to 100%. Two of the internal isolates showed a range of antagonism against M. roseus and E. coli from 1.25 cm ±0.07 cm to 2.35 cm ±0.07 cm and 0.4 cm ±0.00 cm to 2.05 cm ±0.21 cm, respectively. Currently, we are testing these isolates against ESKAPE pathogens and using 16S rRNA sequencing and Biolog ® to further characterize them and their potential as a source of novel antibiotics or bioactive compounds.

Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

Biogenic sulfur recovery from sulfate-laden antibiotic production wastewater using a single-chamber up-flow bioelectrochemical reactor

The high-concentration sulfate (SO42−) in the antibiotic production wastewater hinders the anerobic methanogenic process and also proposes possible environmental risk. In this study, a novel single-chamber up-flow anaerobic bioelectrochemical reactor (UBER) was designed to realize simultaneous SO42− removal and elemental sulfur (S0) recovery. With the carbon felt, the cathode was installed underneath and the anode above to meet the different biological niches for sulfate reducing bacteria (SRB) and sulfur oxidizing bacteria (SOB). The bio-anode UBER (B-UBER) demonstrated a much higher average SO42− removal rate (SRR) of 113.2 ± 5.7 mg SO42−−S L−1 d−1 coupled with a S0 production rate (SPR) of 54.4 ± 5.8 mg S0-S L−1 d−1 at the optimal voltage of 0.8 V than that in the abio-anode UBER (control reactor) (SRR = 86.6 ± 13.4 mg SO42−−S L−1 d−1; SPR = 25.5 ± 9.7 mg S0-S L−1 d−1) under long-term operation. A large amount of biogenic S0 (about 72.2 mg g−1 VSS) was recovered in the B-UBER. The bio-anode, dominated by Thiovirga (SOB genus) and Acinetobacter (electrochemically active bacteria genus), exhibited a higher current density, lower overpotential, and lower internal resistance. C-type cytochromes mainly served as the crucial electron transfer mediator for both direct and indirect electron transfer, so that significantly increasing electron transfer capacity and biogenic S0 recovery. The reaction pathways of the sulfur transformation in the B-UBER were hypothesized that SRB utilized acetate as the main electron donor for SO42− reduction in the cathode zone and SOB transferred electrons to the anode or oxygen to produce biogenic S0 in the anode zone. This study proved a new pathway for biogenic S0 recovery and sulfate removal from sulfate-laden antibiotic production wastewater using a well-designed single-chamber bioelectrochemical reactor.

Chronic toxic effects of erythromycin and its photodegradation products on microalgae Chlorella pyrenoidosa

The photodegradation products (PDPs) of antibiotics in the aquatic environment received increasing concern, but their chronic effects on microalgae remain unclear. This study initially focused on examining the acute effects of erythromycin (ERY), then explored the chronic impacts of ERY PDPs on Chlorella pyrenoidosa. ERY of 4.0 − 32 mg/L ERY notably inhibited the cell growth and chlorophyll synthesis. The determined 96 h median effective concentration of ERY to C. pyrenoidosa was 11.78 mg/L. Higher concentrations of ERY induced more serious oxidative damage, antioxidant enzymes alleviated the oxidative stress. 6 PDPs (PDP749, PDP747, PDP719, PDP715, PDP701 and PDP557) were identified in the photodegradation process of ERY. The predicted combined toxicity of PDPs increased in the first 3 h, then decreased. Chronic exposure showed a gradual decreasing inhibition on microalgae growth and chlorophyll content. The acute effect of ERY PDPs manifested as growth stimulation, but the chronic effect manifested as growth inhibition. The malonaldehyde contents decreased with the degradation time of ERY at 7, 14 and 21 d. However, the malonaldehyde contents of ERY PDPs treatments were elevated compared to those in the control group after 21 d. Risk assessment still need to consider the potential toxicity of degradation products under long-term exposure.

Extended spectrum beta-lactamase production and blaCTX-M gene in Escherichia coli and Klebsiella pneumoniae causing urinary tract infection at a tertiary care hospital in Nepal

Background and objective: Urinary tract infections (UTIs) are the most common bacterial infections where Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the predominating pathogens. These pathogens have a high rate of antibiotic resistance and exhibit the production of extended-spectrum beta-lactamase (ESBL). This study investigated the antibiotic resistance pattern and ESBL production of E. coli and K. pneumoniae isolated from patients with UTIs attending a tertiary care hospital in Nepal by both phenotypic and genotypic techniques. Materials and methods: A cross-sectional study was performed where 4664 mid-stream urine specimens from suspected UTI cases were cultured. Isolated E. coli and K. pneumoniae were subjected to antibiotic susceptibility testing by Kirby Bauer disc diffusion method. Genotypic detection of blaCTX-M gene was performed using polymerase chain reaction (PCR). Results: Out of 4664 urine samples processed, 564 (12.1%) were positive for E. coli (475, 10.2%) and K. pneumonia (89, 1.9%). Out of the total 564 studied samples, 267 (47.3%) were MDR isolates (E. coli: 222, 46.7%; K. pneumoniae: 45, 50.6%) and 96 (17%) were positive for ESBL by double disc confirmatory test. Out of 24 ESBL positive E. coli and 6 K. pneumoniae, 19 (79.2%) and 3 (50%) respectively were positive for blaCTX-M gene. Conclusion: This study indicates high prevalence of MDR and ESBL producing E. coli and K. pneumoniae causing UTIs at an urban hospital setting in Nepal. July 2024; Vol. 18(2):002. DOI: https://doi.org/10.55010/imcjms.18.014 *Correspondence: Gaurab Pandey, Department of Medical Laboratory Science, Nobel College Affiliated to Pokhara University, Kathmandu, Nepal. Email: pandeygaurab67@gmail.com

Effect of commercial probiotics and antibiotics on the growth of Campylobacter isolated from chicken meat in Ho Chi Minh city markets

This study explores the antibiotic susceptibility of Campylobacter, a prominent foodborne pathogen, isolated in Ho Chi Minh city markets and the efficacy of commercial probiotics in inhibiting these bacteria for enhancing food safety and treating Campylobacter infections. Bacteria were isolated from chicken meat in modified coal deoxycholate cefoperazone agar (mCCD), followed by characterization as per standard procedures. Ten isolates with Gram negative, catalase positive and oxidase positive characteristics were collected. Antibiotic susceptibility is ascertained through the determination of the inhibited zone and minimum inhibitory concentration (MIC) of five distinct antibiotics against Campylobacter on Muller Hinton agar plates, culminating in a comprehensive assessment after a 24-hour incubation duration. The antibiotic susceptibility results underscore substantial diversity in Campylobacter isolates among meat samples, thereby accentuating discernible distinctions among the various antibiotic products. The research also evaluated the suitability of 5 commercial probiotic products (re-named as A, B, C, D, and E for fair assessment) by examining their impact on the growth of Campylobacter colonies. The antimicrobial effect of probiotics against Campylobacter is assessed using the agar well diffusion assay and co-culture method. We obtained consistent results from two methods, indicating no variation in Campylobacter species among meat samples but significant variations among probiotic products. The outcomes of this research provide valuable insights into the antimicrobial potential of each probiotic and antibiotic on Campylobacter, informing recommendations for food hygiene practices and underscoring the role of both probiotics and antibiotics in combating Campylobacter infections.

Isolation and Characterization of Antibiotic producing Lysobacter

Isolation and Characterization of Antibiotic producing Lysobacter

Antagonistic Behavior of Streptomyces chartreuse against Pathogenic Bacteria in Ricinus communis L.

ABSTRACT: Antibiotics are a crucial tool in modern medicine and have saved countless lives by effectively treating a wide range of bacterial infections. The microbial antibiotic have several biotechnological applications viz. agriculture, pharmaceuticals, food preservation, animal nutritions. The diverse array of applications and the various roles of bioactive metabolites produced by Actinomycetes have sparked a growing interest in the exploration of unique and unprecedented Actinomycetes strains. The Actinomycetes from soil ecosystem, marine ecosystem, rhizosphere of plant roots are also known to secrete novel antibiotics. In this context, the main objective of this research is to isolate and screen Actinomycetes strains that are capable of producing highly potent culturable secondary metabolites with novel antibacterial properties. These metabolites can potentially serve as biocontrol agents against Xanthomonas infections in Ricinus communis L., offering uncommon and innovative applications within the field of agriculture. All the Actinomycetes isolates were isolated from Mehsana regions of Gujarat an area of over 4,401 km2, with wide microbial diversity and can serve as a source for promising antibiotics producers. 7 rhizospheric soil samples were collected from various region sites viz. Ranasan, Mansa, Panchot, Gozariya, Kansa, Langhnaj, and Kherva. Total 76 antibiotic producing Actinomycetes isolates were obtained in Primary Screening. Based on the results of primary screening, potential morphologically diverse 3 isolates were selected for antibiotic production in liquid medium. FTIR analysis of three samples revealed distinct bands in the spectra. Sample-1 exhibited O-H (1347 cm^-1) and C-N (1191 cm^-1) groups. Sample-2 displayed O-H (3462 cm^-1), C-O (1043 cm^-1), and C=O (1736 cm^-1) groups. Sample-3 showcased O-H (3466 cm^-1), C=O (1737 cm^-1), C-N (1232 cm^-1), and C-O (1043 cm^-1) groups, providing valuable insights into their chemical compositions. The isolate BNPA72 gave best antibiotic production and was identified as Streptomyces chartreusis by 16 s rRNA gene sequencing method. The isolate Streptomyces chartreusis BNPA72 was able to inhibit the plant pathogen Xanthomonas, hence categorized as Biocontrol agents.

Polypeptide Antibiotic Produced by a Thermo-Halophilic Bacterium from Pria Laot Sabang 76 Isolate

Extremophiles are known to produce antibiotics. This study aimed to isolate and purify polypeptide antibiotics from Pria Laot Sabang (PLS) 76, a bacterium isolated from an undersea fumarole. The antibiotic production was done by cultivating PLS 76 in a 2.5% TSB liquid medium and incubated at 70°C, 150 rpm for 112 hours. Ninhydrin assay on the crude supernatant after centrifugation indicated that the PLS 76 produced a polypeptide antibiotic. The supernatant was partitioned using methanol. The antibiotic activity was tested using the Kirby-Bauer method, and the inhibition zone was about 12 mm against Escherichia coli and Staphylococcus aureus. The methanol fraction was purified further by Preparative Thin Layer Chromatography and produced several fractions. Fractions with the same Rf were pooled and subjected to the ninhydrin assay. Fraction A, which gave a positive result after the ninhydrin assay, showed an inhibition zone of only 7 mm against E. coli and S. aureus. The Quadrupole Time-of-Flight (QTOF) Mass Spectrometry data indicated that the antibiotic's structure was similar to Polymyxin B2, an antibiotic of a polypeptide group.

Non-pathogenic Heyndrickxia coagulans (Bacillus coagulans) 29-2E inhibits the virulence of pathogenic Salmonella Typhimurium by quorum-sensing regulation

Bacteria produce and release small signal molecules, autoinducers, as an indicator of their cell density. The system, called a quorum-sensing (QS) system, is used to control not only virulence factors but also antibiotic production, sporulation, competence, and biofilm formation in bacteria. Different from antibiotics, QS inhibitors are expected to specifically repress the virulence factors in pathogenic bacteria without inhibiting growth or bactericidal effects. Therefore, since QS inhibitors have little risk of antibiotic-resistant bacteria emergence, they have been proposed as promising anti-bacterial agents. In the present study, we aimed to find new QS inhibitors that prohibit the signaling cascade of autoinducer 3 (AI-3) recognized by a QseCB two-component system that regulates some virulence factors of pathogens, such as enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica subsp. enterica serovar Typhimurium. We have established the method for QS-inhibitor screening using a newly constructed plasmid pLES-AQSA. E.coli DH5α transformed with the pLES-AQSA can produce β-galactosidase that converts 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal) into blue pigment (5-bromo-4-chloro-indoxyl) under the control of the QseCB system. By screening, Heyndrickxia coagulans (formerly Bacillus coagulans) 29-2E was found to produce an exopolysaccharide (EPS)-like water-soluble polymer that prohibits QseCB-mediated β-galactosidase production without antibacterial activities. Further, the simultaneous injection of the 29-2E strain significantly improves the survival rate of Salmonella Typhimurium-infected silkworm larvae (from 0% to 83.3%), suggesting that the substance may be a promising inhibitor against the virulence of pathogens without risk of the emergence of antibiotic-resistant bacteria.

Phylogeny of Transferable Oxazolidinone Resistance Genes and Homologs.

Oxazolidinone resistance, especially transmissible resistance, is a major public health concern, and the origin of this resistance mechanism is not yet resolved. This study aims to delve into the phylogenetic origin of the transmissible oxazolidinone resistance mechanisms conferring cross-resistance to other drugs of human and veterinary importance. The amino acid sequences of the five cfr ribosomal methylases and optrA and poxtA were used as queries in searches against 219,549 bacterial proteomes in the NCBI RefSeq database. Hits with >40% amino acid identity and >80% query coverage were aligned, and phylogenetic trees were reconstructed. All five cfr genes yielded highly similar trees, with rlmN housekeeping ribosomal methylases located basal to the sister groups of S-adenosyl-methionine-dependent methyltransferases from various Deltaproteobacteria and Actinomycetia, including antibiotic-producing Streptomyces species, and the monophyletic group of cfr genes. The basal branches of the latter contained paenibacilli and other soil bacteria; they then could be split into the clades [cfr(C):cfr(E)] and [[cfr:cfr(B)]:cfr(D)], always with different Bacillaceae in their stems. Lachnospiraceae were encountered in the basal branches of both optrA and poxtA trees. The ultimate origin of the cfr genes is the rlmN housekeeping ribosomal methylases, which evolved into a suicide-avoiding methylase in antibiotic producers; a soil organism (Lachnospiraceae, Paenibacilli) probably acted as a transfer organism into pathogenic bacteria. In the case of optrA, the porcine pathogenic Streptococcus suis was present in all branches, while the proteins closest to poxtA originated from Clostridia.

Comparative genomics reveals insight into the phylogeny and habitat adaptation of novel Amycolatopsis species, an endophytic actinomycete associated with scab lesions on potato tubers.

A novel endophytic actinomycete, strain MEP2-6T, was isolated from scab tissues of potato tubers collected from Mae Fag Mai Sub-district, San Sai District, Chiang Mai Province, Thailand. Strain MEP2-6T is a gram-positive filamentous bacteria characterized by meso-diaminopimelic acid in cell wall peptidoglycan and arabinose, galactose, glucose, and ribose in whole-cell hydrolysates. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and hydroxy-phosphatidylethanolamine were the major phospholipids, of which MK-9(H6) was the predominant menaquinone, whereas iso-C16:0 and iso-C15:0 were the major cellular fatty acids. The genome of the strain was 10,277,369 bp in size with a G + C content of 71.7%. The 16S rRNA gene phylogenetic and core phylogenomic analyses revealed that strain MEP2-6T was closely related to Amycolatopsis lexingtonensis NRRL B-24131T (99.4%), A. pretoriensis DSM 44654T (99.3%), and A. eburnea GLM-1T (98.9%). Notably, strain MEP2-6T displayed 91.7%, 91.8%, and 87% ANIb and 49%, 48.8%, and 35.4% dDDH to A. lexingtonensis DSM 44653T (=NRRL B-24131T), A. eburnea GLM-1T, and A. pretoriensis DSM 44654T, respectively. Based on phenotypic, chemotaxonomic, and genomic data, strain MEP2-6T could be officially assigned to a novel species within the genus Amycolatopsis, for which the name Amycolatopsis solani sp. nov. has been proposed. The type of strain is MEP2-6T (=JCM 36309T = TBRC 17632T = NBRC 116395T). Amycolatopsis solani MEP2-6T was strongly proven to be a non-phytopathogen of potato scab disease because stunting of seedlings and necrotic lesions on potato tuber slices were not observed, and there were no core biosynthetic genes associated with the BGCs of phytotoxin-inducing scab lesions. Furthermore, comparative genomics can provide a better understanding of the genetic mechanisms that enable A. solani MEP2-6T to adapt to the plant endosphere. Importantly, the strain smBGCs accommodated 33 smBGCs encoded for several bioactive compounds, which could be beneficially applied in the fields of agriculture and medicine. Consequently, strain MEP2-6T is a promising candidate as a novel biocontrol agent and antibiotic producer.

A quorum-sensing regulatory cascade for siderophore-mediated iron homeostasis in Chromobacterium violaceum.

Iron is a transition metal used as a cofactor in many biochemical reactions. In bacteria, iron homeostasis involves Fur-mediated de-repression of iron uptake systems, such as the iron-chelating compounds siderophores. In this work, we identified and characterized novel regulatory systems that control siderophores in the environmental opportunistic pathogen Chromobacterium violaceum. Screening of a 10,000-transposon mutant library for siderophore halos identified seven possible regulatory systems involved in siderophore-mediated iron homeostasis in C. violaceum. Further characterization revealed a regulatory cascade that controls siderophores involving the transcription factor VitR acting upstream of the quorum-sensing (QS) system CviIR. Mutation of the regulator VitR led to an increase in siderophore halos, and a decrease in biofilm, violacein, and protease production. We determined that these effects occurred due to VitR-dependent de-repression of vioS. Increased VioS leads to direct inhibition of the CviR regulator by protein-protein interaction. Indeed, insertion mutations in cviR and null mutations of cviI and cviR led to an increase of siderophore halos. RNA-seq of the cviI and cviR mutants revealed that CviR regulates CviI-dependent and CviI-independent regulons. Classical QS-dependent processes (violacein, proteases, and antibiotics) were activated at high cell density by both CviI and CviR. However, genes related to iron homeostasis and many other processes were regulated by CviR but not CviI, suggesting that CviR acts without its canonical CviI autoinducer. Our data revealed a complex regulatory cascade involving QS that controls siderophore-mediated iron homeostasis in C. violaceum.IMPORTANCEThe iron-chelating compounds siderophores play a major role in bacterial iron acquisition. Here, we employed a genetic screen to identify novel siderophore regulatory systems in Chromobacterium violaceum, an opportunistic human pathogen. Many mutants with increased siderophore halos had transposon insertions in genes encoding transcription factors, including a novel regulator called VitR, and CviR, the regulator of the quorum-sensing (QS) system CviIR. We found that VitR is upstream in the pathway and acts as a dedicated repressor of vioS, which encodes a direct CviR-inhibitory protein. Indeed, all QS-related phenotypes of a vitR mutant were rescued in a vitRvioS mutant. At high cell density, CviIR activated classical QS-dependent processes (violacein, proteases, and antibiotics production). However, genes related to iron homeostasis and type-III and type-VI secretion systems were regulated by CviR in a CviI- or cell density-independent manner. Our data unveil a complex regulatory cascade integrating QS and siderophores in C. violaceum.

Complete genome sequence of a novel Prescottella sp. R16 isolate from deep-sea sediments in the western Pacific.

Prescottella, a distinct genus separate from Rhodococcus, has garnered attention for its adaptability and ecological versatility. In this study, a Gram-stain positive and ovoid-rod shaped the actinobacterium strain R16 was isolated from deep-sea sediment (with a depth of 6,310m) in the Western Pacific. On the basis of 16S rRNA gene sequence analysis, average nucleotide identity and phylogenomic analysis, strain R16 clearly represents a novel species within the genus Prescottella. Genomic analyses indicate Prescottella sp. R16 contains a circular chromosome of 4,531,251 bp with an average GC content of 68.9%, 4,208 protein-coding genes, 51 tRNA genes, and 12 rRNA operons. Additionally, four CRISPRs and 24 genomic islands are also identified. The presence of rich categories related to catalytic activity, membrane part and metabolic process highlights their involvement in cellular component, biological process, and molecular function. The genome sequence of strain R16 also revealed the presence of 13 putative biosynthetic gene clusters for secondary metabolites, including those for ε-Poly-L-lysine, ectoine, heterobactin, isorenieratene and corynecin, suggesting its potential for antibiotic production and warranting further exploration.

Pyoluteorin regulates the biosynthesis of 2,4-DAPG through the TetR family transcription factor PhlH in Pseudomonas protegens Pf-5.

Soil and rhizosphere bacteria act as a rich source of secondary metabolites, effectively fighting against a diverse array of pathogens. Certain Pseudomonas species harbor biosynthetic gene clusters for producing both pyoluteorin and 2,4-diacetylphloroglucinol (2,4-DAPG), which are polyketides that exhibit highly similar antimicrobial spectrum against bacteria and fungi or oomycete. A complex cross talk exists between pyoluteorin and 2,4-DAPG biosynthesis, and production of 2,4-DAPG was strongly repressed by pyoluteorin, yet the underlying mechanism is still elusive. In this study, we find that the TetR family transcription factor PhlH is involved in the cross talk between pyoluteorin and 2,4-DAPG biosynthesis. PhlH binds to a palindromic sequence within the promoter of phlG (PphlG), which encodes a C-C bond hydrolase responsible for degrading 2,4-DAPG. As a signaling molecule, pyoluteorin disrupts the PhlH-PphlG complex by binding to PhlH, leading to decreased levels of 2,4-DAPG. Proteomics data suggest that pyoluteorin regulates multiple physiological processes including fatty acid biosynthesis and transportation of taurine, siderophore, and amino acids. Our work not only reveals a novel mechanism of cross talk between pyoluteorin and 2,4-DAPG biosynthesis, but also highlights pyoluteorin's role as a messenger in the complex communication network of Pseudomonas.IMPORTANCEAntibiosis serves as a crucial defense mechanism for microbes against invasive bacteria and resource competition. These bacteria typically orchestrate the production of multiple antibiotics in a coordinated fashion, wherein the synthesis of one antibiotic inhibits the generation of another. This strategic coordination allows the bacterium to focus its resources on producing the most advantageous antibiotic under specific circumstances. However, the underlying mechanisms of distinct antibiotic production in bacterial cells remain largely elusive. In this study, we reveal that the TetR family transcription factor PhlH detects the secondary metabolite pyoluteorin and mediates the cross talk between pyoluteorin and 2,4-DAPG biosynthesis in the biocontrol strain Pseudomonas protegens Pf-5. These findings hold promise for future research, potentially informing the manipulation of these systems to enhance the effectiveness of biocontrol agents.

Multifactorial genetic control and magnesium levels govern the production of a Streptomyces antibiotic with unusual cell density dependence.

Streptomyces bacteria are renowned both for their antibiotic production capabilities and for their cryptic metabolic potential. Their metabolic repertoire is subject to stringent genetic control, with many of the associated biosynthetic gene clusters being repressed by the conserved nucleoid-associated protein Lsr2. In an effort to stimulate new antibiotic production in wild Streptomyces isolates, we leveraged the activity of an Lsr2 knockdown construct and successfully enhanced antibiotic production in the wild Streptomyces isolate WAC07094. We determined that this new activity stemmed from increased levels of the angucycline-like family member saquayamycin. Saquayamycin has both antibiotic and anti-cancer activities, and intriguingly, beyond Lsr2-mediated repression, we found saquayamycin production was also suppressed at high density on solid or in liquid growth media; its levels were greatest in low-density cultures. This density-dependent control was exerted at the level of the cluster-situated regulatory gene sqnR and was mediated in part through the activity of the PhoRP two-component regulatory system, where deleting phoRP led to both constitutive antibiotic production and sqnR expression. This suggests that PhoP functions to repress the expression of sqnR at high cell density. We further discovered that magnesium supplementation could alleviate this density dependence, although its action was independent of PhoP. Finally, we revealed that the nitrogen-responsive regulators GlnR and AfsQ1 could relieve the repression exerted by Lsr2 and PhoP. Intriguingly, we found that this low density-dependent production of saquayamycin was not unique to WAC07094; saquayamycin production by another wild isolate also exhibited low-density activation, suggesting that this spatial control may serve an important ecological function in their native environments.IMPORTANCEStreptomyces specialized metabolic gene clusters are subject to complex regulation, and their products are frequently not observed under standard laboratory growth conditions. For the wild Streptomyces isolate WAC07094, production of the angucycline-family compound saquayamycin is subject to a unique constellation of control factors. Notably, it is produced primarily at low cell density, in contrast to the high cell density production typical of most antibiotics. This unusual density dependence is conserved in other saquayamycin producers and is driven by the pathway-specific regulator SqnR, whose expression is influenced by both nutritional and genetic elements. Collectively, this work provides new insights into an intricate regulatory system governing antibiotic production and indicates there may be benefits to including low-density cultures in antibiotic screening platforms.

EXPLORING THE SOIL-ASSOCIATED BACTERIAL MICROBIOME OF COFFEE PLANTATIONS IN DIFFERENT REGIONS OF COLOMBIA: A METABARCODING APPROACH

<p><strong><span lang="EN-US">Introduction:</span></strong><span lang="EN-US"> Coffee is one of the leading tropical crops produced worldwide. Colombia ranks third in coffee production in the world. Microorganisms associated with coffee plants can have many biotechnological applications, such as plant growth promotion and biological control. <strong>Objective:</strong> To describe the bacteria present in the soil associated with coffee cultivation. This was done using a bulk sequencing or 16S rRNA metabarcoding approach. <strong>Methodology:</strong> Soil samples were collected to analyze their microbiome from three different departments of Colombia (Cauca, Risaralda and Magdalena). The plants were of different ages, and the crop management was different (conventional and organic). Subsequently, an analysis was carried out using Qiime2 to describe the communities associated with coffee cultivation and soil chemical properties. <strong>Results:</strong> Some important genera were identified, such as <em>Janthinobacterium</em>, <em>Bacillus</em>, <em>Actinomadura</em> and <em>Actinoallomurus</em>. These genera can be used as plant growth promoters, organic matter transformers, producers of antibiotics and metabolites with potential biotechnological applications. <strong>Implications:</strong> The study presented in this manuscript describes the communities associated with the soil in coffee cultivation. However, a much more complex approach could be through metagenomics, where the bacterial communities and the functions of; however, this vision is up to ten times higher in cost. <strong>Conclusion:</strong> Through the results obtained, it is concluded that the studies carried out through metabarcoding help to understand the composition of the microorganisms associated with coffee and, with this, try to elucidate the functions of the associated microorganisms.</span></p>

Expanding the Biosynthetic Toolbox: The Potential and Challenges of In Vitro Type II Polyketide Synthase Research

Type II polyketide synthase (PKS) systems are a rich source of structurally diverse polycyclic aromatic compounds with clinically relevant antibiotic and chemotherapeutic properties. The enzymes responsible for synthesizing the polyketide core, known collectively as the minimal cassette, hold potential for applications in synthetic biology. The minimal cassette provides polyketides of different chain lengths, which interact with other enzymes that are responsible for the varied cyclization patterns. Additionally, the type II PKS enzyme clusters offer a wide repertoire of tailoring enzymes for oxidations, glycosylations, cyclizations, and rearrangements. This review begins with the variety of chemical space accessible with type II PKS systems including the recently discovered highly reducing variants that produce polyalkenes instead of the archetypical polyketide motif. The main discussion analyzes the previous approaches with an emphasis on further research that is needed to characterize the minimal cassette enzymes in vitro. Finally, the potential type II PKS systems hold the potential to offer new tools in biocatalysis and synthetic biology, particularly in the production of novel antibiotics and biofuels.