Formulation of Anti Acne Sheet Mask from Bandotan Leaf Extract (Ageratum conyzoides L.) against Propionibacterium acnes

Acne can occur due to increased sebum excretion, inflammation of the skin triggered by the bacteria Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus. The purpose of this study was to determine the formulation of sheet mask preparations of bandotan leaf extract (Ageratum conyzoides L.) in inhibiting the growth of Propionibacterium acnes bacteria. This type of research is an experimental study which include plant identification, making ethanol extract of bandotan leaves, making sheet mask formulations, evaluating the characteristics of the preparation and testing antibacterial activity using disc paper method using Mueller Hinton Agar media. The extract was carried out by maceration using 70% ethanol as a solvent. Testing the effectiveness of antibacterial by measuring the diameter of the inhibition zone, after that the data were analyzed using the One Way ANOVA statistical test. The results of the evaluation of the preparation showed that the preparation was homogenous, with pH ranging from 4.5 to 6.5 and the preparation did not cause irritation on volunteers skin. The results of the inhibition zone measurements showed that the inhibition zones at 2.5% concentration is 4.7mm, 5% (6.83mm), 7.5% (10.2mm) and positive control (20.57mm). This means that the higher the concentration, the larger the diameter of the inhibition zone obtained. The conclusion in this study is bandotan leaf extract (Ageratum conyzoides L.) can be formulated into anti acne sheet mask which is stable during storage with a strong inhibitory power at 7.5% concentration, which is 10.2 mm. Keywords: Ageratum conyzoides L., bandotan leaf, Propionibacterium acnes, sheet mask

ISOLATION METHODS FOR MOLECULAR DETECTION AND ANTIBIOTIC RESISTANCE PATTERN OF CAMPYLOBACTER SPP IN LAYER CHICKENS

This study was conducted to compare two culture methods for the isolation of Campylobacter spp from commercial layer chickens and subsequently confirmed by Polymerase Chain Reaction assays (PCR). Furthermore, the antimicrobial resistance profiles of PCR positive Campylobacter isolates were determined.Cloacal swab samples (550) from chickens randomly selected from five poultry farms in the four geographical zones in Ogun State were cultured for Campylobacter using modified charcoal Cefoperazone deoxycholate agar (MCCDA) and an improved culture method involving Preston broth pre-enrichment and subsequent subculture on Mueller Hinton agar with Campylobacter growth supplements. Putative isolates were later confirmed by PCR assay and sequencing analysis.Other isolates that grew on MCCDA and confirmed by sequencing analysis are Enterococcus faecalis, Escherichis coli, Comamonas kerstli and Pseudomonas aeroginusa . The antibiotic resistant profile of all the isolates were evaluated genotypically for resistance genes to tetracyclines (tetO), multiclasses (cmeB), aminoglycosides (aphA-3-1) and β-lactams (Blaoxa-61) using multiplex PCR (mPCR), and phenotypically for chlortetracycline, tylosin, streptomycin, ciprofloxacin and erythromycin resistance by microbroth dilution method which correspond to the antibiotic resistance genes. The apparent prevalence of Campylobacter was 16.8% by MCCDA while none of the isolates was positive to PCR. Meanwhile, prevalence rate of 26% was obtained using Preston broth pre-enrichment and Mueller Hinton agar with Campylobacter growth supplements, of which 11/50 (22%) of the isolates was confirmed positive by PCR. Genotypic characterization of PCR positive isolates showed 10/11(90%) were C. coli, 1/11(10%) other Campylobacter species and 0% C. jejuni. All the isolates carried both tetO and cmeB resistant genes. The results of minimum inhibitory concentration presented all PCR positive isolates had resistance of 10/10(100%), 9/10(90%), 6/10(60%), 9/10(90%), and 8/10(80%) to tetracycline, ciprofloxacin, erythromycin, spectinomycin and tylosin respectively. In addition, all isolates carried multiple resistance to most antibiotics tested which are commonly used in poultry practice in Nigeria. Campylobacter spp in the study areas showed diverse genotypic characteristics, and gene mediated multidrug resistance.

Isolation, Identification and Antibiotic Susceptibility of Escherichia coli Isolated from Raw Meat from Modake and Ile-Ife, Osun State, Nigeria

The study reported isolation, identification and antibiotic susceptibility of Escherichia coli isolated from raw meat from Modakeke and Ile-ife, Osun State, Nigeria, with the view to determining the antibiogram profiling of the bacterial isolates. In this study, five samples of fresh meat were collected from different abattoirs in Ile-Ife and Modakeke, Osun State. Isolates of Escherichia coli were isolated, identified morphologically based on their growth on nutrient agar and subjected to antibiotic susceptibility test on Mueller Hinton agar. The mean microbial load from the meat samples ranged from 8.85 x 102cfu/ml to 5.77 x 104cfu/ml. A total of 69 E. coli isolates were recovered from the meat sampled. All the isolates appeared cream, translucent, entire, convex, circular, smooth and glistering. The isolates were identified as Gram negative rods, non-motile, lactose fermenters, positive for indole test and negative for citrate utilization test. All the E. coli isolates were resistant to augmentin, ceftriazone, nitrofurantoin and gentamycin. 98.55% of E. coli isolated was resistant to amoxillin and the least resistant was recorded in ofloxacin (8.70%). However, 91.30% of the E. coli isolates was sensitive to ofloxacin, 81.16% to ciprofloxacin and 36.23% to pefloxacin while none was sensitive to augmentin, ceftriazone, nitrofurantoin and gentamycin. A total of 19 different multiple antibiotic resistance patterns were observed among the isolates. Thirty isolates (43.48%) showed multiple antibiotic resistance to 5 and 10 different antibiotic types each. The study concluded that occurrence of E. coli infection is high in the study area with high level of multiple antibiotic resistance.

1287. Double Disk Diffusion Study to Evaluate the Synergistic Effect Between Cefiderocol and Ceftazidime-Avibactam Against Cefiderocol-Non-susceptible Acinetobacter baumannii

Background Cefiderocol (CFDC), a siderophore cephalosporin, has broad coverage of Gram-negative bacteria and has been approved for clinical use in USA and Europe. The SIDERO-WT surveillance studies showed that CFDC shows >95% susceptibility against Acinetobacter baumannii. Against many of CFDC non-susceptible isolates, most of which had blaPER gene, the combination use of avibactam significantly decreased the MIC by broth microdilution (BMD). In this study, we evaluated the appropriate methodology to evaluate the synergistic effect by disk diffusion studies. Methods The susceptibility testing was conducted as recommended by the CLSI using CFDC non-susceptible isolates (MIC of >4 µg/mL based on CLSI breakpoint). The MIC by BMD was determined using iron-depleted cation-adjusted Mueller-Hinton broth, in the presence or absence of 4 µg/mL of avibactam. The disk diffusion was evaluated using Mueller-Hinton agar, and the synergy was evaluated by using disk stacking methods. For disk stacking methods, CFDC disk was placed on agar on which bacterial suspension of 0.5 McFarland units was spread, then the ceftazidime-avibactam (CZA) disks was stacked on the top, followed by adding a drop (30 µL) of saline on the stacked disks. As an alternative method, CZA was immersed in saline for 1 second instead of adding a drop of saline, followed by the stacking on the top. The disk zone size was determined after 24-hour incubation at 37°C. Results Against blaPER-positive A. baumannii which showed >64 µg/mL MIC of CFDC and CZA, CFDC MIC decreased to 0.25 µg/mL in the presence of avibactam. The disk diffusion methods also showed isolates resistant to CFDC and CZA and showed susceptiblilty disk zone to CFDC by stacking both disks. On the other hand, against blaNDM-positive A. baumannii which showed 64 µg/mL MIC of CFDC and CZA, the disk diffusion methods showed resistance even when stacking both disks. Against multiple isolates, the MIC of CFDC without or with avibactam was correlated well with the disk zone produced by CFDC disk alone or stacked with CZA disks, respectively (Figure). Conclusion The synergistic effect between CFDC and avibactam by BMD methods could be detected by disk stacking methodology using CFDC and CZA disks. Disclosures Yoshinori Yamano, PhD, Shionogi (Employee) Miki Takemura, MS, SHIONOGI & CO., LTD. (Employee) Roger Echols, MD, Shionogi (Consultant) Christopher Longshaw, PhD, Shionogi (Employee)

TWO ALTERNATIVE METHODS FOR ANAEROBIC CULTIVATION OF BACTEROIDES FRAGILIS ATCC 25285 COMPARED TO THE EFFICIENCY OF CULTIVATION IN A GAS PACK SYSTEM

Cultivation of anaerobes is not a routine procedure. In most cases, it is carried out in reference laboratories with the help of special equipment, including an anaerobic chamber or anaerobic jar with Gas pack system. There are some older methods so-called the Candlejar system and the Fortner principle. The aim of the study is to compare two alternative methods for anaerobic cultivation of Bacteroides fragilis ATCC 25285 with cultivation by Gas Pack system, which is a widely used and well studied method. We made anaerobic cultivation using the GasPack system, Candle jar and Fortner principle. The bacteria were seed in two different types of agar media - suitable for anaerobic cultivation Wilkins-Chalgren agar and Mueller-Hinton agar, useful for antibiotic sensitivity testing. The results of our study showed that in all bacterial cultures, B. fragilis ATCC 25285 demonstrated heavy growth. The Candle jar system and Fortner principle were found to be a sensitive and cost-effective alternative that might be used in resource-limited settings.

In vitro synergistic activity of aztreonam (AZT) plus novel and old β-lactamase inhibitor combinations against metallo-β-lactamase-producing AZT-resistant Enterobacterales

The emergence of metallo-β-lactamase (MBL)-producing Enterobacterales , mainly New Delhi metallo-β-lactamase (NDM), represents a clinical threat due to the limited therapeutic alternatives. Aztreonam (AZT) is stable to MBLs, but most MBL-producing Enterobacterales isolates usually co-harbour other β-lactamases that confer resistance to AZT and, consequently, its use is restricted in these isolates. We compared the ability of sulbactam (SUL), tazobactam (TAZ), clavulanic acid (CLA) and avibactam (AVI) to restore the AZT activity in MBL-producing AZT-resistant Enterobacterales isolates. A collection of 64 NDM-producing AZT-resistant Enterobacterales from five hospitals in Buenos Aires city, Argentina, were studied during the period July–December 2020. MICs were determined using the agar dilution method with Mueller–Hinton agar according to Clinical and Laboratory Standards Institute (CLSI) recommendations. AVI, SUL and TAZ were used at a fixed concentration of 4 mg l−1, whereas CLA was used at a fixed concentration of 2 mg l−1. A screening method based on disc diffusion to evaluate this synergy was also conducted. Detection of bla KPC, bla OXA, bla NDM, bla VIM, bla CTXM-1, bla PER-2 and bla CIT was performed by PCR. The AZT-AVI combination restored the AZT activity in 98.4 % of AZT-resistant strains, whereas CLA, TAZ and SUL did so in 70.3, 15.6 and 12.5 %, respectively, in isolates co-harbouring extended-spectrum β-lactamases, but were inactive in isolates harbouring AmpC-type enzymes and/or KPC. The synergy screening test showed an excellent negative predictive value to confirm the absence of synergy, but positive results should be confirmed by a quantitative method. The excellent in vitro performance of the AZT-CLA combination represents a much more economical alternative to AZT-AVI, which could be of use in the treatment of MBL-producing, AZT-resistant Enterobacterales .

Evaluation of cefiderocol activity for Gram-negative bacteria and performance of cefiderocol disk diffusion testing using multiple brands of Mueller Hinton Agar

Cefidericol is a cephalosporin-siderophore antibiotic for the treatment of multidrug resistant Gram-negative bacteria. Similar to other cephalosporin antibiotics, the lethal mechanism of action is due to inhibition of penicillin binding proteins leading to lysis of the bacteria. However, unlike previously developed antibiotics, the siderophore portion of cefidericol is able to bind iron and then be actively transported into the periplasmic space. To ascertain the feasibility of cefidericol antibiotic susceptibility testing in the Barnes-Jewish Clinical Microbiology Laboratory, we collected a cohort of multidrug Enterobacteriacae (5 Enterobacter cloace, 8 Escherichia coli, 12 Klebsiella pneumoniae), Pseudomonas aeruginosa (n=23), Stenotrophomonas maltophila (n=24), and Acinetobacter baumannii (n=25). We evaluated activity of cefidericol on these strains, and the performance of disk diffusion using three different brands of Mueller-Hinton Agar (BD, Hardy, and Remel). The reference method for comparison was an FDA-cleared broth microdilution panel containing cefidericol (ThermoFisher Scientific). Using CLSI breakpoints, we found that disk diffusion with BD agar had 96% categorical agreement for Enterobacterales, 100% for P. aeruginosa, 92% for A. baumannii, 96% for S. maltophila. We found that Hardy had 96% categorical agreement for Enterobacterales, 92% for P. aeruginosa, 92% for A. baumannii, 96% for S. maltophila. Finally, we found that Hardy had 96% categorical agreement for Enterobacterales, 92% for P. aeruginosa, 92% for A. baumannii, 96% for S. maltophila. Minor errors on any media never exceed 4% and there were no very major errors. Resistance to cefidericol within our cohort of selected antibiotic resistant bacteria was rare, one E. coli isolate and two P. aeruginosa isolates had minimal inhibitory concentrations (MICs) > 32 μg/mL. The highest MICs for one isolate of A. baumannii and one isolate S. maltophila was 8 μg/mL and 4 μg/mL, respectively, both of which were intermediate. There was no difference in the distribution of zone disk diffusion diameter for A. baumannii or Enterobacterales. However, there was a significant difference in the distribution of zone disk diffusion diameters for P. aeruginosa and S. maltophila on BD vs Hardy agar. The median for P. aeruginosa on BD is 25 mm while it is 29 mm on Hardy. The trend for S. maltophila is the opposite as the median for BD was 31.5 mm and 28.5 mm for Hardy. Use of FDA vs CLSI vs EUCAST breakpoints significantly changes outcome of susceptibility testing for broth microdilution and disk diffusion. As one example for broth microdilution of A. baumannii, we had one isolate intermediate using CLSI breakpoints, 4 resistant using EUCAST breakpoints, and 4 resistant and 3 intermediate isolates using FDA breakpoints. Our work demonstrates that cefedericol testing can be performed in a routine format, with certain organismal differences on Mueller-Hinton agar, and that different interpretative criteria significantly change outcomes.

Susceptibilities of invasive Neisseria meningitidis strains to agents used for prophylaxis and to penicillin G

Antimicrobial susceptibility of 50 Neisseria meningitidis strains detected in Nova Scotia between 2004 and 2018 was determined. The isolates were cultured from sites that might prompt chemoprophylaxis (27 blood, 18 cerebrospinal fluid [CSF], 3 CSF–blood, and 2 conjunctiva). Minimal inhibitory concentrations (MICs) were determined to azithromycin, ciprofloxacin, minocycline, rifampin, trimethoprim–sulfamethoxazole, and penicillin G, using a diffusion gradient strip on Mueller–Hinton agar with 5% sheep blood in 5% CO2 for 20–24 hours. All isolates remained susceptible to azithromycin, ciprofloxacin, minocycline, and rifampin, but there was 26% resistance to trimethoprim–sulfamethoxazole. There was a rise in penicillin MIC of the isolates over the study period.

Antimicrobial effects of an organosilane-based disinfectant

Organosilane-based disinfectants have been purported to impart lasting antimicrobial properties when applied to a surface. The implications being dramatic reduction in pathogen transmission via fomites and therefore of great public health importance as a means to limit the spread of outbreaks such as COVID-19. We conducted experiments in our laboratory to assess the efficacy of one such product, which reported anti-microbial protection of up to 120 days, as an operational report for our hospital. 1) The organosilane product was applied on a Mueller Hinton agar plate and left overnight underneath an airconditioned vent to promote mould growth. 2) The organosilane product was applied on a Blood Agar Plate (BAP) that was inoculated with E. Coli and observed over 24 hours. 3) Lastly, the organosilane coating was applied onto plastic plates to simulate hospital ward surfaces. Simulated pathogens (suspension of oral commensals) were spread across the plastic plates, swabbed at 1 hour, 8 hours and 24 hours and then incubated for 24 hours. In all 3 experiments, there was no significant difference in microbial growth between the control and intervention arms. Although our experiments did not assess different methods of application nor effect on different materials applied, the lack of demonstrable effects of the organosilane product calls for caution against widespread use of a product whose claims are unverified.

Occurrence of Multidrug-Resistant Staphylococcus aureus among Humans, Rodents, Chickens, and Household Soils in Karatu, Northern Tanzania

We conducted this study to investigate the isolation frequency and phenotypic antibiotic resistance pattern of Staphylococcus aureus isolated from rodents, chickens, humans, and household soils. Specimens were plated onto mannitol salt agar (Oxoid, Basingstoke, UK) and incubated aerobically at 37 °C for 24 h. Presumptive colonies of S. aureus were subjected to Gram staining, as well as catalase, deoxyribonuclease (DNAse), and coagulase tests for identification. Antibiotic susceptibility testing was performed by using the Kirby–Bauer disc diffusion method on Mueller–Hinton agar (Oxoid, Basingstoke, UK). The antibiotics tested were tetracycline (30 μg), erythromycin (15 μg), gentamicin (10 μg), ciprofloxacin (5 μg), clindamycin (2 μg), and amoxicillin-clavulanate (20 μg/10 μg). The S. aureus strain American Type Culture Collection (ATCC) 25,923 was used as the standard organism. We found that 483 out of 956 (50.2%) samples were positive for S. aureus. The isolation frequencies varied significantly between samples sources, being 52.1%, 66.5%, 74.3%, and 24.5%, respectively, in chickens, humans, rodents, and soil samples (p < 0.001). S. aureus isolates had high resistance against clindamycin (51.0%), erythromycin (50.9%), and tetracycline (62.5%). The overall prevalence of multidrug-resistant (MDR) S. aureus isolates was 30.2%, with 8.7% resistant to at least four different classes of antibiotics.