Isolation, Serovar Identification, and Antimicrobial Susceptibility of Avibacterium paragallinarum from Chickens in China from 2019 to 2020

Infectious coryza is an acute infectious respiratory disease in chickens that is caused by Avibacterium paragallinarum (A. paragallinarum). Infectious coryza has major economic effects due to decreased egg production in growing birds and slowed growth in broilers. In this study, we isolated and identified 40 strains of A. paragallinarum from chickens that showed typical clinical signs of coryza in part of China from 2019 to 2020. Using a hemagglutination-inhibition test, 11 isolates were identified as serovar A, 10 isolates were identified as serovar B, and 19 isolates were identified as serovar C. Antimicrobial sensitivity tests showed that high minimum inhibitory concentration (MIC) values were encountered for compounds sulfamethoxine sodium and oxytetracycline hydrochloride. Especially, of the 40 A. paragallinarum isolates, 30% had an MIC value of compound sulfamethoxine sodium of 64 μg/mL, 10% of 128 μg/mL, and 15% of 256 μg/mL. For oxytetracycline hydrochloride, 85% of isolates showed MIC values of 64 μg/mL or more. Excitingly, the MIC values of β-lactamase (amoxicillin, ampicillin, and ceftiofur) were low, with 77.5%, 70%, and 92.5% of isolates having an MIC value of ≤1 μg/mL, respectively. Our results may provide a reference for the treatment of infectious coryza.

Evaluation of Antimicrobial Effects of Four Selected Marine Macroalgae from Iskenderun Bay

In the present study, the seaweeds belong to Phaeophyaceae (Cystoseira elegans Sauvageau 1912, Cystoseira amentacea (C.Agardh) Bory 1832, Padina crassa Yamada 1931) and Florideophyceae (Corallina elongata J.Ellis & Solander 1786) collected from nearby Iskenderun-Turkey of Mediterranean Sea were detected for their antimicrobial activities against seven bacterial strains (Escherichia coli ATCC 35218, Bacillus cereus NRRL B-371, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 13883, Salmonella typhimurium ATCC 14028, Proteus vulgaris RSKK 96029). The antimicrobial activities were expressed as minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). According to the results obtained from MICs and MBCs values of the extracts on pathogenic microorganisms were between 0.78 to 50 mg/mL. The lowest MICs and MBCs values were recorded for C. elegans extract against B. cereus with a MIC value of 0.78 mg/mL. These results suggest that secondary metabolites of brown and red algae are important sources that could be used as broad spectrum of biological and pharmaceutical activities.

Scalarane Sesterterpenoids with Antibacterial and Anti-Proliferative Activities from the Mushroom Neonothopanus nambi

Seven undescribed scalarane sesterterpenoids, nambiscalaranes B–H (1–7), together with two known compounds, nambiscalarane (8) and aurisin A (9) were isolated from the cultured mycelium of the luminescent mushroom Neonothopanus nambi. Their structures were elucidated by thorough analysis of their 1D and 2D NMR spectroscopic data. The absolute configurations of 1–8 were determined by electronic circular dichroism (ECD) calculations and optical rotation measurements. The isolated sesterterpenoids were evaluated against A549, HT29, HeLa, and HCT-116 cancer cell lines, and against five bacterial strains. Compounds 3, 5, and 7 showed strong cytotoxicity against HCT-116 cell line, with IC50 values ranging from 13.41 to 16.53 µM, and showed no cytotoxicity towards Vero cells. Moreover, compound 8 inhibited the growth of Bacillus subtilis with a MIC value of 8 µg/mL, which was equivalent to the MIC value of the standard kanamycin.

The Synthesis and Evaluation of Heterocycles as Anti-Tuberculosis Agents

<p>Tuberculosis (TB) is the leading cause of death from a single infectious agent, Mycobacterium tuberculosis (Mtb), worldwide. Currently, the efficacy of TB treatment regimens has declined due to the rise in antibacterial resistance and the shortage of new TB drugs. Thus, much effort has been spent in anti-tuberculosis drug development and in identifying new therapeutic targets against Mtb. One such target is NADH dehydrogenase-II (NDH-II), an essential enzyme in the mycobacterial electron transport chain that is not present in mammalian cells. In this thesis, four classes of heterocyclic compounds that have the potential to target NDH-II and their evaluation as anti-tubercular agents, are described. An overview of TB drug development and NDH-II as a promising target for TB drugs are described in Chapter 1. In Chapters 2 and 3, the potential of anti-tubercular drugs based on the quinolinequinone (QQ) scaffold is described. QQs have previously shown promise as TB drugs by activating NDH-II to overproduce harmful reactive oxygen species leading to bacterial cell death. Chapter 2 describes the total synthesis of the QQ natural products ascidiathiazone A and ascidiathiazone B, and derivatives thereof, using a synthetic route that allows for high divergency and the efficient synthesis of the natural products and their intermediates. To this end, the first total synthesis of ascidiathiazone B is reported, as is the identification of ascidiathiazone A as a promising anti-tuberculosis drug with an MIC of 1.6 μM against Mtb. Insight into the ability of a representative quinone, 7-chloro-6-chloroethylamino-2-methyl-QQ, to increase NDH-II activity is also described. In Chapter 3, the syntheses of thirty-two simplified QQs with different functional groups at the 6- and 7-positions of the QQ scaffold are described. These compounds were screened against Mtb, with the lead compound from this library, 7-chloro-6-propargylamino-QQ, exhibiting an MIC of 8 μM against Mtb. Structure-activity data revealed diminished biological activity for QQs bearing tertiary amines, as compared to those with secondary amines, suggesting that the presence of a hydrogen bond donor at the 6- and 7-positions of QQs may play a critical role in antimycobacterial activity. In Chapter 4, the synthesis and anti-mycobacterial activity of chromonyl-pyrimidines is presented. Chromonyl-pyrimidines have a structural resemblance to quinolinyl pyrimidines, a class of known NDH-II inhibitors and anti-TB agents. Chromones have shown promise as TB drugs, though they have not yet been reported to bind NDH-II. Despite this, chromonyl-pyrimidines contain a ketone functionality that may be able to bind the quinone binding site. For the first eleven-member library of chromonyl pyrimidines synthesised, all but two of the compounds exhibited inhibitory activity against Mtb, however, the growth inhibition was modest (MIC = 36-684 M). Accordingly, a second generation of chromonyl pyrimidines was synthesised, which included six compounds with improved potency against Mtb – all with an MIC value of 12.5 μM. The activity of these chromonyl pyrimidines was attributed to the presence of aromatic rings both on the pyrimidine and the chromone scaffolds, though changes to the electronic properties of the aryl groups, i.e. the incorporations of electron-withdrawing and electron-donating groups, did not affect inhibitory activity. Finally, in Chapter 5, a library of phthalazinones and pyrimidinyl-phthalazinones with anti-tubercular activity is described. While phthalazinones have not yet been extensively explored as anti-mycobacterial agents, the phthalazinone scaffold has the potential to act as an uncoupler. Uncouplers are typically weak acids or bases that act on the electron transport chain by dissipating the proton motive force and ultimately preventing the generation of ATP. In Mtb, this uncoupling process is detrimental and leads to cell death. Phthalazinones are weakly basic and, due to their bicyclic ketone-bearing motif, has the potential to bind NDH-II at the proposed Q-site. Accordingly, a series of phthalazinones was synthesised to investigate their anti-tubercular activity and uncoupling activity. From the library of phthalazinones, N-tert-butyl- and nitro-substituted phthalazinones elicited high inhibitory activity, both with an MIC value of 3 μM. Of particular note among the pyrimidinyl-phthalazinones was the 4-fluorophenyl-pyrimidinyl-N-heptyl phthalazinone, which showed high potency against Mtb with an MIC of 1.6 μM. Further biological studies showed that some phthalazinones increased the rate of NADH oxidation in mycobacteria, which could be a result of uncoupling activity, while a number of pyrimidinyl-phthalazinones decreased NADH oxidation rates. These mechanistic results indicated that the two classes of compounds may have different modes of inhibition.</p>

The Synthesis and Evaluation of Heterocycles as Anti-Tuberculosis Agents

<p>Tuberculosis (TB) is the leading cause of death from a single infectious agent, Mycobacterium tuberculosis (Mtb), worldwide. Currently, the efficacy of TB treatment regimens has declined due to the rise in antibacterial resistance and the shortage of new TB drugs. Thus, much effort has been spent in anti-tuberculosis drug development and in identifying new therapeutic targets against Mtb. One such target is NADH dehydrogenase-II (NDH-II), an essential enzyme in the mycobacterial electron transport chain that is not present in mammalian cells. In this thesis, four classes of heterocyclic compounds that have the potential to target NDH-II and their evaluation as anti-tubercular agents, are described. An overview of TB drug development and NDH-II as a promising target for TB drugs are described in Chapter 1. In Chapters 2 and 3, the potential of anti-tubercular drugs based on the quinolinequinone (QQ) scaffold is described. QQs have previously shown promise as TB drugs by activating NDH-II to overproduce harmful reactive oxygen species leading to bacterial cell death. Chapter 2 describes the total synthesis of the QQ natural products ascidiathiazone A and ascidiathiazone B, and derivatives thereof, using a synthetic route that allows for high divergency and the efficient synthesis of the natural products and their intermediates. To this end, the first total synthesis of ascidiathiazone B is reported, as is the identification of ascidiathiazone A as a promising anti-tuberculosis drug with an MIC of 1.6 μM against Mtb. Insight into the ability of a representative quinone, 7-chloro-6-chloroethylamino-2-methyl-QQ, to increase NDH-II activity is also described. In Chapter 3, the syntheses of thirty-two simplified QQs with different functional groups at the 6- and 7-positions of the QQ scaffold are described. These compounds were screened against Mtb, with the lead compound from this library, 7-chloro-6-propargylamino-QQ, exhibiting an MIC of 8 μM against Mtb. Structure-activity data revealed diminished biological activity for QQs bearing tertiary amines, as compared to those with secondary amines, suggesting that the presence of a hydrogen bond donor at the 6- and 7-positions of QQs may play a critical role in antimycobacterial activity. In Chapter 4, the synthesis and anti-mycobacterial activity of chromonyl-pyrimidines is presented. Chromonyl-pyrimidines have a structural resemblance to quinolinyl pyrimidines, a class of known NDH-II inhibitors and anti-TB agents. Chromones have shown promise as TB drugs, though they have not yet been reported to bind NDH-II. Despite this, chromonyl-pyrimidines contain a ketone functionality that may be able to bind the quinone binding site. For the first eleven-member library of chromonyl pyrimidines synthesised, all but two of the compounds exhibited inhibitory activity against Mtb, however, the growth inhibition was modest (MIC = 36-684 M). Accordingly, a second generation of chromonyl pyrimidines was synthesised, which included six compounds with improved potency against Mtb – all with an MIC value of 12.5 μM. The activity of these chromonyl pyrimidines was attributed to the presence of aromatic rings both on the pyrimidine and the chromone scaffolds, though changes to the electronic properties of the aryl groups, i.e. the incorporations of electron-withdrawing and electron-donating groups, did not affect inhibitory activity. Finally, in Chapter 5, a library of phthalazinones and pyrimidinyl-phthalazinones with anti-tubercular activity is described. While phthalazinones have not yet been extensively explored as anti-mycobacterial agents, the phthalazinone scaffold has the potential to act as an uncoupler. Uncouplers are typically weak acids or bases that act on the electron transport chain by dissipating the proton motive force and ultimately preventing the generation of ATP. In Mtb, this uncoupling process is detrimental and leads to cell death. Phthalazinones are weakly basic and, due to their bicyclic ketone-bearing motif, has the potential to bind NDH-II at the proposed Q-site. Accordingly, a series of phthalazinones was synthesised to investigate their anti-tubercular activity and uncoupling activity. From the library of phthalazinones, N-tert-butyl- and nitro-substituted phthalazinones elicited high inhibitory activity, both with an MIC value of 3 μM. Of particular note among the pyrimidinyl-phthalazinones was the 4-fluorophenyl-pyrimidinyl-N-heptyl phthalazinone, which showed high potency against Mtb with an MIC of 1.6 μM. Further biological studies showed that some phthalazinones increased the rate of NADH oxidation in mycobacteria, which could be a result of uncoupling activity, while a number of pyrimidinyl-phthalazinones decreased NADH oxidation rates. These mechanistic results indicated that the two classes of compounds may have different modes of inhibition.</p>

Chemical Composition and Biological Activities of Eugenia caryophyllata Thunb Essential Oil

Aims: In this study the antibacterial and antioxidant activities of the essential oil of Eugenia caryophyllata were investigated. Study design: The study contains determination of the chemical composition of the essential oil of E. caryophyllata and the in vitro evaluation of the antibacterial and antioxidant activity of this oil. Place and Duration of Study: The study was carried out at the laboratory of research on local animal products of Ibn-Khaldoun University, Tiaret, Algeria during the period from December 2020 to March 2021. Methodology: The essential oil composition was characterized by gas chromatography/mass spectrophotometrical analyses. The antibacterial activity of this oil was tested against four pathogenic bacteria: Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 33862, Bacillus cereus ATCC 11778 by using disc diffusion method and agar incorporation method to determine the Minimum Inhibitory Concentration (MIC) of the tested oil. The antioxidant activity was evaluated by using DPPH radical scavenging, hydrogen peroxide scavenging assays and ferric reducing antioxidant power (FRAP) assay. Results: Our results have shown a greater antibacterial effect of E. caryophyllata essential oil against all the bacterial tested strains with inhibition zone diameters varied from 17.5 to 20.5 mm and minimal inhibition concentration (MIC) ranged between 0.8 μl / ml and 4.4 μl / ml, B. cereus and S. aureus are the most sensitive species with a MIC value of 0.8 μl / ml, however P. aeruginosa is the most resistant species with a MIC value of 4.4 μl / ml. The result of the antioxidant effect showed that the essential oil of E. caryophyllata is a powerful antioxidant that expresses a higher antioxidant activity than the standard antioxidants: gallic acid, vitamin C and BHT. Conclusion: The obtained results suggest that the essential oil of E. caryophyllata has a strong antibacterial and antioxidant effect and it may be an alternative natural source medicine to prevent and treat many diseases caused by pathogenic bacteria and oxidative stress.

1208. Omadacycline In Vitro Activity Against Bacillus Anthracis

Background Bacillus anthracis, the etiological agent of anthrax, is one of the agents most likely to be used in a biologic attack. Omadacycline previously has demonstrated potent in vitro and in vivo activity against B. anthracis. This project evaluated the in vitro activity of omadacycline against a larger set of B. anthracis strains across two laboratories. Methods Methods: Antibiotic susceptibility testing followed Clinical Laboratory Standard Institute methods against a collection of 53 B. anthracis strains at the University of Florida (UF) and 50 B. anthracis strains at MRIGlobal, representing human and animal isolates from North America, Africa, Europe, Asia, and Australia. Minimum inhibitory concentrations (MICs) for omadacycline and comparators at both sites (doxycycline, ciprofloxacin, levofloxacin, moxifloxacin) were determined by broth microdilution. Results Results: In the UF study, omadacycline demonstrated an MIC50 of 0.015 mg/L and an MIC90 of 0.03 mg/L against B. anthracis. Omadacycline MIC values were equal to or lower than doxycycline. In the MRIGlobal study, omadacycline demonstrated an MIC50 of 0.06 mg/L and an MIC90 of 0.06 mg/L (Table 1). All comparator MIC values were within ranges previously observed against these strains. Against a ciprofloxacin-resistant strain (MIC = 2 mg/L), omadacycline had an MIC value of 0.015 mg/L; against a doxycycline-resistant strain (MIC = 4 mg/L), omadacycline had an MIC value of 0.06 mg/L. Reproducibility was observed between the 2 laboratories for omadacycline in vitro activity against B. anthracis (Table 2). Table 1. MIC Concentration Summary for Omadacycline and Comparators Against B. anthracis Strains Table 2. Reproducibility of Omadacycline in Vitro Activity Against B. anthracis Strains Conclusion Based on the in vitro activity in both studies, omadacycline has the potential to be effective in treating anthrax infection. Reproducibility of omadacycline in vitro activity against B. anthracis was observed at 2 independent study sites. Disclosures Alisa W. Serio, PhD, Paratek Pharmaceuticals, Inc. (Employee, Shareholder) Diane M. Anastasiou, BA, Paratek Pharmaceuticals, Inc. (Consultant)

647. Investigation of Heteroresistance Among Klebsiella pneumoniae (KP) Inner Colonies (IC) Observed During Fosfomycin Disk Diffusion (DD) Testing

Background During fosfomycin DD testing, the frequent occurrence of non-susceptible IC within the zone of inhibition of susceptible isolates has been noted. The Clinical & Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) have contradicting recommendations on how IC should be interpreted; CLSI recommends considering IC when interpreting DD results whereas EUCAST recommends ignoring them. This study sought to identify the susceptibility of these IC and to understand whether heteroresistance contributes to the appearance of IC during fosfomycin DD. Methods This study included a convenience sample of 71 KP clinical isolates from 3 United States locations. During DD testing, 58 (81.7%) of these isolates displayed at least one IC. Broth microdilution (BMD) minimal inhibitory concentration (MIC) testing, using extrapolated CLSI Escherichia coli breakpoints, was performed on a subset (n=32) of the IC in duplicate for comparison to the corresponding parent MIC values. This was followed by a modified disk elution screening test for heteroresistance to compare the frequency of low level resistance (LLR) and high level resistance (HLR) between the susceptible isolates that produced resistant IC (n=6) and those that did not produce any IC (n=3). Results The MIC range for the IC isolates (128 to &gt; 1024 μg/mL) increased as compared to the parent isolates (&lt; 2 to &gt; 256 μg/mL) and MIC50/90 increased from the parent (128/ &gt; 256 μg/mL) to IC (1024/ &gt; 1024 μg/mL) isolates. All IC isolates had a resistant MIC value vs. 46.5% of parent isolates, and over 90% of IC isolates had an MIC at least 2 dilutions higher than their corresponding parent isolate. Heteroresistance screening found all tested isolates to be positive for LLR, and 8 of 9 positive for HLR, while the one HLR-negative isolate was IC-producing. Conclusion IC were frequent during fosfomycin DD testing and were commonly more resistant than their corresponding KP parent isolates. A small subset of these isolates tested via a modified disk elution test displayed either LLR or HLR regardless of the absence of IC. These results call for further investigation among a larger isolate set to understand what mechanisms are responsible for the frequency of IC and their increased fosfomycin resistance. Disclosures All Authors: No reported disclosures

Bioaccumulation and detoxification of trivalent arsenic by Achromobacter xylosoxidans BHW-15 and electrochemical detection of its transformation efficiency

Arsenotrophic bacteria play an essential role in lowering arsenic contamination by converting toxic arsenite [As (III)] to less toxic and less bio-accumulative arsenate [As (V)]. The current study focused on the qualitative and electrocatalytic detection of the arsenite oxidation potential of an arsenite-oxidizing bacteria A. xylosoxidans BHW-15 (retrieved from As-contaminated tube well water), which could significantly contribute to arsenic detoxification, accumulation, and immobilization while also providing a scientific foundation for future electrochemical sensor development. The minimum inhibitory concentration (MIC) value for the bacteria was 15 mM As (III). Scanning Electron Microscopy (SEM) investigation validated its intracellular As uptake capacity and demonstrated a substantial association with the MIC value. During the stationary phase, the strain’s As (III) transformation efficiency was 0.0224 mM/h. Molecular analysis by real-time qPCR showed arsenite oxidase (aioA) gene expression increased 1.6-fold in the presence of As (III) compared to the untreated cells. The immobilized whole-cell also showed As (III) conversion up to 18 days. To analyze the electrochemical oxidation in water, we developed a modified GCE/P-Arg/ErGO-AuNPs electrode, which successfully sensed and quantified conversion of As (III) into As (V) by accepting electrons; implying a functional As oxidase enzyme activity in the cells. To the best of our knowledge, this is the first report on the electrochemical observation of the As-transformation mechanism with Achromobactersp. Furthermore, the current work highlighted that our isolate might be employed as a promising candidate for arsenic bioremediation, and information acquired from this study may be helpful to open a new window for the development of a cost-effective, eco-friendly biosensor for arsenic species detection in the future.

Australian Gonococcal Surveillance Programme Annual Report, 2020

The Australian Gonococcal Surveillance Programme (AGSP), established in 1981, has continuously monitored antimicrobial resistance in clinical isolates of Neisseria gonorrhoeae for more than 40 years. In 2020, a total of 7,222 clinical isolates of gonococci from patients in the public and private sectors, in all jurisdictions, were tested for in vitro antimicrobial susceptibility by standardised methods. Current treatment recommendations for gonorrhoea, for the majority of Australia, continues to be dual therapy with ceftriaxone and azithromycin. In 2020, decreased susceptibility (DS) to ceftriaxone (minimum inhibitory concentration [MIC] value ≥ 0.06 mg/L) was found nationally in 0.9% of isolates. There was one isolate, reported from Victoria in 2020, that was resistant to ceftriaxone (MIC value ≥ 0.25 mg/L). Resistance to azithromycin (MIC value ≥ 1.0 mg/L) was found nationally in 3.9% of N. gonorrhoeae isolates, continuing a downward trend observed and reported since 2017. Isolates with high-level resistance to azithromycin (MIC value ≥ 256 mg/L) are identified sporadically in Australia; in 2020, there was one such isolate reported in Queensland. In 2020, penicillin resistance was found in 27% of gonococcal isolates nationally, and ciprofloxacin resistance in 36%; however, there is considerable variation by jurisdiction. In some remote settings, penicillin resistance remains low, and this drug continues to be recommended as part of an empiric therapy strategy. In 2020, in remote Northern Territory, no penicillin resistance was reported, and in remote Western Australia 5/116 of gonococcal isolates (4.3%) were penicillin resistant. There was one ciprofloxacin-resistant isolate reported from remote Northern Territory, and ciprofloxacin resistance rates remain comparatively low in remote Western Australia (4/116; 3.4%).