Agar Dilution Test Research Articles

Novel patterns in the molecular epidemiology of KPC-producing Klebsiella pneumoniae in Tucumán, Argentina.

In Argentina, there has been an abrupt increase in KPC-2-producing Klebsiella pneumoniae (K. pneumoniae). Tucumán is a multi-border area, so the rapid dissemination of carbapenem-resistant K. pneumoniae is a clinically relevant problem for the region. This study aimed to investigate the epidemiological and molecular patterns of KPC-producing K. pneumoniae clinical isolates collected from different hospitals in Tucumán. Carbapenem-resistant K. pneumoniae strains were sequentially and uniquely collected during two time periods. Antibiotic susceptibility was determined by the automated Vitex 2® system and using the standard agar dilution test. Multilocus sequence typing and pulsed-field electrophoresis were used for epidemiological analysis. The genetic structures around blaKPC and the encoding genes of extended-spectrum β-lactamases were detected by polymerase chain reaction and sequencing. Plasmids were analysed by conjugation and using the plasmid relaxase gene-typing method. All 37 isolates were multidrug resistant, and theblaKPC-2 gene was confirmed in all of them. In 17 isolates (45.9%), the blaCTX-M-2 gene was also amplified, as well as blaSHV-2 in five isolates (13.5%) and blaCTX-M-2/blaSHV-2 in four isolates (10.8%). The molecular epidemiology of the blaKPC-2 gene has resulted in it being associated with an IncL/M transferable plasmid disseminating in various sequence types (STs) (ST17, ST556, ST342, ST147, ST461, ST65, ST15 and ST70), and in a new genetic environment with a 764-bp deletion in the ISKpn7-blaKPC region. These findings contribute to the understanding of the great diversity of the blaKPC-2-carrying genetic platforms.

Decreased susceptibility to chlorhexidine and distribution of qacA/B genes among coagulase-negative Staphylococcus clinical samples

BackgroundHealthcare-associated infection (HAI) is a major public health problem. As a form of prevention and control, preparations of chlorhexidine are used extensively; however, the reduction of susceptibility to chlorhexidine has been reported. The aim of this study was to investigate the susceptibility to chlorhexidine and the distribution of the qacA/B genes in 211 clinical isolates of coagulase-negative Staphylococci (CoNS).MethodsCoNS were identified by conventional biochemical tests. Antimicrobial susceptibility was tested by disk-diffusion. Minimum inhibitory concentration (MIC) of chlorhexidine was determined by agar dilution test; detection of the qacA/B and mecA genes were evaluated by PCR.ResultsThe most frequently isolated species were S. epidermidis, S. hominis hominis, S. auricularis, and S. haemolyticus, respectively. The strains presented a multidrug resistance profile of 87%, including methicillin resistance. Reduced susceptibility to chlorhexidine was observed in 31%. The qacA/B genes were detected in samples resistant (32/32) and susceptible (17/32) to chlorhexidine. The vast majority (94%) of the samples with reduced susceptibility to chlorhexidine were multidrug resistant.ConclusionsOur results show that qacA/B genes are not restricted to strains expressing chlorhexidine resistance. Further studies are needed to understand how the expression of these genes occurs.

Recent Increase in the Incidence of TEM-135 β-Lactamase-harboring Neisseria gonorrhoeae in Korea.

BackgroundWe investigated the molecular epidemiological characteristics and antimicrobial susceptibility pattern of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates to monitor the change in distribution of blaTEM in Korea.MethodsWe collected 804 PPNG isolates from diverse hospitals and clinics mainly located in Seoul, Korea, over a period of 11 years (2005–2015). Isolate susceptibility to seven antimicrobials was determined using the agar dilution test. The molecular epidemiological characteristics of the isolates were determined by Sanger sequencing of blaTEM, N. gonorrhoeae multiantigen sequence typing (NG-MAST) and plasmid typing.ResultsAmong 72 fully sequenced PPNG isolates, sixteen (22.2%) possessed TEM-135. All TEM-135 isolates had a common silent mutation (c.18C>T), which was previously unreported. We observed a pattern of continuous increase in the number of TEM-135 isolates since 2012. The median and 90% minimum inhibitory concentration of azithromycin were substantially lower in the TEM-135 group than in the non-PPNG and TEM-1 groups. All TEM-135 isolates showed different NG-MAST types and predominantly harbored Toronto/Rio (75%) plasmids. A comprehensive comparative analysis of PPNG with TEM-135 according to NG-MAST, plasmid type, and year of isolation revealed a wide distribution.ConclusionsThe proportion of TEM-135 PPNG has continuously increased since 2012, in association with clonal spread. The difference at position 18 of the TEM-135 sequence can be interpreted as the existence of multiple clonal complexes. The possibility that TEM-135 was acquired via foreign plasmids requires careful follow-up and continuous monitoring of TEM-135 to ascertain whether it constitutes a step towards evolutionary change.

RdxA, frxA, and efflux pump in metronidazole-resistant Helicobacter pylori: Their relation to clinical outcomes.

rdxA and frxA mutations and enhancement of efflux pump have been suggested as the cause of metronidazole resistance in Helicobacter pylori. This study was performed to investigate the resistance mechanisms related to clinical eradication outcome, and to examine direct involvement of hefA in metronidazole-resistant isolates with intact rdxA and frxA. A total of 53 H.pylori-positive patients who were treated with metronidazole-containing sequential or quadruple therapy from 2011 to 2015 were enrolled. The metronidazole susceptibility of H.pylori isolates was examined by agar dilution test. Mutations in rdxA and frxA, were analyzed with DNA sequencing, and impact of hefA on metronidazole resistance was examined with quantitative real-time reverse transcription polymerase chain reaction, knockout and genetic complementation test for hefA. Seven mutation types of rdxA and/or frxA were found in H.pylori isolated from non-eradicated subjects. rdxA mutation was associated with eradication failure (P=0.002), and nonsense mutation in rdxA reduced eradication efficacy (P=0.009). hefA expression was significantly higher in resistant isolates (P<0.001), especially in rdxA(-)frxA(-) as compared to rdxA(+)frxA(+) (P=0.027). Resistant isolates with no mutation in rdxA and frxA became susceptible after hefA knockout. Genetic complementation for hefA recovered metronidazole resistance in all of three hefA knockout mutants. These results suggest that rdxA mutations play a critical role in metronidazole resistance as well as the outcomes of eradication therapy. In addition, hefA seems to be directly involved in metronidazole resistance, which explains the resistance in clinical isolates with intact rdxA and frxA.

Impact of amoxicillin resistance on the efficacy of amoxicillin-containing regimens for Helicobacter pylori eradication: analysis of five randomized trials.

The impact of amoxicillin resistance on the efficacy of regimens containing amoxicillin for Helicobacter pylori eradication remains unknown. To investigate whether the efficacy of an amoxicillin-containing regimen is affected by amoxicillin resistance and to identify the optimal breakpoint for amoxicillin resistance. This was a pooled analysis of five randomized trials conducted in Taiwan from 2007 to 2016. Patients who received amoxicillin-containing regimens were recruited. MICs were determined by agar dilution testing. Meta-analysis was performed to assess the risk ratio of eradication failure in amoxicillin-resistant strains compared with susceptible strains of seven different regimens. We performed further the pooled analysis and logistic regression in patients treated with clarithromycin triple therapy to identify the optimal breakpoint for amoxicillin resistance. A total of 2339 patients with available amoxicillin MICs were enrolled. Meta-analysis showed that the presence of amoxicillin resistance was consistently associated with increased risk of treatment failure of amoxicillin-containing regimens at different breakpoints (risk ratio: 1.41, 95% CI 1.12-1.78, P = 0.004 when the cut-off was 0.5 mg/L). The heterogeneity was low (I2 = 0%, P = 0.615). Pooled analysis also showed that amoxicillin resistance was an independent risk factor for treatment failure of clarithromycin triple therapy at different breakpoints. The best correlation was observed when the breakpoint of amoxicillin resistance was ≥0.125 mg/L (kappa coefficient 0.298), at which the resistance rate was 11.1% (110 of 990). The efficacies of amoxicillin-containing regimens are affected by amoxicillin resistance and the optimal breakpoint MIC is ≥ 0.125 mg/L.

Prevalence and Sensitivity of Bacilli and Pseudomonas in the Newborn's Oral Cavity.

The aim of this study was to isolate Enterobacteria and Pseudomonas from the oral cavity of hospitalized newborns (NB) and determine their prevalence and the sensitivity profile to most commonly used antibiotics for this age group. Samples from the oral cavity of NB from 24 to 48 h age were collected using swabs. The samples were inoculated on MacConkey agar, incubated and the colonies counted and identified. For each strain, the minimum inhibitory concentration (MIC) was determined using agar dilution test. Tests for enterobacteria producing extended spectrumβ-lactamases (ESBL) were performed using agar diffusion. Descriptive statistics was used for data analysis. Two of the isolated strains were submitted to the susceptibility test in biofilm. Of the collected samples, 8% presented Enterobacteria (mean of 6,141 CFU/mL) and no Pseudomona species was isolated. Positive samples were from NB in accommodation set or in the NB nursery. Enterobacter was the most prevalent genus and some strains were resistant to ampicillin, gentamicin and cephalothin. No ESBL strain was detected. Microorganisms in biofilms were resistant to all antibiotics, with concentrations four times higher than MIC. The presence of enterobacteria in the oral cavity of newborns, especially some strains resistant to normally used antibiotics, warns to the need for care to avoid the early colonization of this niche and the occurrence of a possible hospital infection in this age group.

Synthesis and characterization of acrylamide-based copolymeric hydrogel–silver composites: Antimicrobial activities and inhibition kinetics against E. coli

ABSTRACTA series of acrylamide (AAm)-based hydrogels containing acrylic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), and vinyl imidazole (VI) comonomers were prepared by free radical polymerization. Silver nanoparticles were loaded to hydrogel systems through in situ reduction of silver nitrate in the presence of sodium borohydride as a reducing agent. The synthesized hydrogels and their composites were characterized using FTIR, scanning electron microscopy, EDX, and EDX-mapping. The antimicrobial activity of hydrogel–silver composite was determined using well agar and broth dilution tests. In the first stage, four different hydrogel–silver composites were evaluated against six different microorganisms using the well agar technique. The most effective hydrogel–silver composite among all tested was poly(AAm-co-VI-co-AMPS)-Ag, while the most sensitive and resistant microorganisms among all tested were Staphylococcus cerevisiae and S. aureus, respectively. Poly(AAm-co-VI-co-AMPS)-Ag composite was used in modeling the inhibition kinetic of Escherichia coli. The present study displays that hydrogel–silver composite has considerable antimicrobial activity, which deserves further investigation for use in clinic application and industrial processing.

Cluster of Neisseria gonorrhoeae Isolates With High-level Azithromycin Resistance and Decreased Ceftriaxone Susceptibility, Hawaii, 2016.

The Centers for Disease Control and Prevention (CDC) currently recommends dual therapy with ceftriaxone and azithromycin for gonorrhea to ensure effective treatment and slow emergence of antimicrobial resistance. Since 2013, the prevalence of reduced azithromycin susceptibility increased in the United States; however, these strains were highly susceptible to cephalosporins. We identified a cluster of Neisseria gonorrhoeae isolates with high-level azithromycin resistance, several of which also demonstrated decreased ceftriaxone susceptibility. Eight N. gonorrhoeae isolates collected from 7 patients on Oahu, Hawaii, seen 21 April 2016 through 10 May 2016 underwent routine Etest antimicrobial susceptibility testing by the Hawaii Department of Health. All demonstrated elevated azithromycin minimum inhibitory concentrations (MICs) >256 μg/mL and elevated ceftriaxone MICs (≥0.125 μg/mL). Isolates were sent to the University of Washington and CDC for confirmatory agar dilution testing; sequence data were sent to CDC for analysis. All patients were interviewed and treated, and when possible, partners were interviewed, tested, and treated. All isolates had azithromycin MICs >16 µg/mL and 5 had ceftriaxone MICs = 0.125 µg/mL by agar dilution. All isolates were β-lactamase positive and were resistant to penicillin, tetracycline, and ciprofloxacin. Genomic analysis revealed genetic relatedness. No patients reported recent travel or antibiotic use, and no male patients reported male sex partners. All patients were successfully treated. This cluster of genetically related gonococcal isolates with decreased ceftriaxone susceptibility and high-level azithromycin resistance may bring the threat of treatment failure in the United States with the current recommended dual therapy one step closer.

New insights on the antibacterial efficacy of miconazole in vitro.

Miconazole is a broad-spectrum antifungal used in topical preparations. In the present investigation the minimal inhibitory concentration (MIC) of miconazole for eighty wild type strains of gram-positive and gram-negative bacteria isolated from infected skin lesions was assessed using a modified agar dilution test (adapted to CLSI, Clinical Laboratory Standards Institute). 14 ATCC reference strains served as controls. Miconazole was found efficacious against gram-positive aerobic bacteria (n=62 species), the MICs against Staphylococcus (S.) aureus, S. spp., Streptococcus spp. und Enterococcus spp. ranged between 0.78 and 6.25μg/mL. Interestingly, there were no differences in susceptibility between methicillin-susceptible (MSSA, 3) methicillin-resistant (MRSA, 6) and fusidic acid-resistant (FRSA, 2) S.aureus isolates. Strains of Streptococcus pyogenes (A-streptococci) (8) were found to be slightly more sensitive (0.78-1.563μg/mL), while for gram-negative bacteria, no efficacy was found within the concentrations tested (MIC >200μg/mL). In conclusion, for the gram-positive aerobic bacteria the MICs of miconazole were found within a range which is much lower than the concentration of miconazole used in topical preparations (2%). Thus topically applied miconazole might be a therapeutic option in skin infections especially caused by gram-positive bacteria even by those strains which are resistant to antibiotics.

Epidemiological characteristics of blaNDM-1 gene in Enterobacteriaceae strains circulating in China during 2013 to 2014

Objective To investigate the epidemiological characteristics of blaNDM-1 gene (encoding New Delhi metallo-β-lactamase 1) in Enterobacteriaceae strains circulating in China during July 2013 to June 2014. Methods A total of 2 577 Enterobacteriaceae strains were collected from 19 Class A tertiary hospitals nationwide by China Antimicrobial Resistance Surveillance Trial (CARST). PCR was used to detect the presence of blaNDM-1 gene in those Enterobacteriaceae strains. For blaNDM-1-positive strains, Analytical Profile Index (API) strips and 16S rRNA analysis were used to identify bacterial genus and species. Antibiotic susceptibilities were assessed by detecting minimum inhibitory concentrations (MIC) against them with two-fold agar dilution test. Multilocus-sequence typing (MLST) and pulsed field gel electrophoresis (PFGE) were performed for homology analysis. Results Among the 2 577 strains, 24 from 8 different provinces and cities carried the blaNDM-1 gene, including 11 Klebsiella pneumoniae strains, 4 Enterobacter cloacae strains, 3 Providencia rettgeri strains, 2 Escherichia coli strains, 2 Citrobacter freundii strains, 1 Klebsielia Oxytoca and 1 Serratia marcescens, and all of them were multidrug-resistant (MDR). ST17 (5, 45.45%) and ST20 (3, 27.27%) were the predominant sequence types of Klebsiella pneumoniae strains. Strains of the same STs were clonally related. PFGE analysis showed that 3 Providencia rettgeri strains isolated in Nanjing belonged to the same clone. Conclusion The positive rate of blaNDM-1 gene in Enterobacteriaceae strains isolated during 2013 to 2014 is 0.93%, indicating a significant increase as compared with that during 2009 to 2010 (0%) and 2011 to 2012 (0.32%). The present study first reports the strain of Serratia marcescens harboring NDM-1 gene in China. NDM-1-producing strains are highly resistant to multiple drugs. Most of the NDM-1-producing Enterobacteriaceae strains were Klebsiella pneumoniae strains (45.83%) warranting special attention as there is a risk of endemic outbreak. Key words: Beta-lactamase NDM-1; Enterobacteriaceae; Epidemiology

Epidemiology and antibiotic sensitivity of Staphylococcus aureus nasal carriage in children in Hungary.

The aim of this study was to assess the Staphylococcus aureus nasal carriage rate in healthy children all over Hungary and to specify some risk factors, the antibiotic resistance patterns of the bacteria, and their genetic relatedness. In total, 878 children (aged 3-6 years) were screened at 21 day-care centers in 16 different cities in Hungary, between February 2009 and December 2011. Samples taken from both nostrils were cultured on blood agar, and suspected S. aureus isolates were identified by β-hemolysis, catalase positivity, clump test, and nucA PCR. Methicillin-resistant strains were screened by mecA and mecC PCR. Antibiotic susceptibility was determined by agar dilution or gradient test strips. Pulsed-field gel electrophoresis was used for genotyping. S. aureus carriage rate was found to be 21.3%, which correlates well with international data. We found no statistically significant correlation between the gender or the sibling status and S. aureus carriage. All isolates were sensitive to oxacillin, trimethoprim-sulfamethoxazole, and mupirocin. The resistance rates for erythromycin, ciprofloxacin, clindamycin, gentamicin, and tetracycline were 7.5%, 0.5%, 1.1%, 3.7%, and 4.3%, respectively. The isolates showed very high genetic diversity. In summary, carried S. aureus isolates are more sensitive to antibiotics compared with clinical isolates in Hungary, and methicillin-resistant S. aureus carriage rate is very low yet.

Inhibition of Helicobacter pylori and Its Associated Urease by Palmatine: Investigation on the Potential Mechanism.

In this paper, we evaluated the anti-Helicobacter pylori activity and the possible inhibitory effect on its associated urease by Palmatine (Pal) from Coptis chinensis, and explored the potential underlying mechanism. Results indicated that Pal exerted inhibitory effect on four tested H. pylori strains (ATCC 43504, NCTC 26695, SS1 and ICDC 111001) by the agar dilution test with minimum inhibitory concentration (MIC) values ranging from 100 to 200 μg/mL under neutral environment (pH 7.4), and from 75 to 100 μg/mL under acidic conditions (pH 5.3), respectively. Pal was observed to significantly inhibit both H. pylori urease (HPU) and jack bean urease (JBU) in a dose-dependent manner, with IC50 values of 0.53 ± 0.01 mM and 0.03 ± 0.00 mM, respectively, as compared with acetohydroxamic acid, a well-known urease inhibitor (0.07 ± 0.01 mM for HPU and 0.02 ± 0.00 mM for JBU, respectively). Kinetic analyses showed that the type of urease inhibition by Pal was noncompetitive for both HPU and JBU. Higher effectiveness of thiol protectors against urease inhibition than the competitive Ni2+ binding inhibitors was observed, indicating the essential role of the active-site sulfhydryl group in the urease inhibition by Pal. DTT reactivation assay indicated that the inhibition on the two ureases was reversible, further supporting that sulfhydryl group should be obligatory for urease inhibition by Pal. Furthermore, molecular docking study indicated that Pal interacted with the important sulfhydryl groups and inhibited the active enzymatic conformation through N-H ∙ π interaction, but did not interact with the active site Ni2+. Taken together, Pal was an effective inhibitor of H. pylori and its urease targeting the sulfhydryl groups, representing a promising candidate as novel urease inhibitor. This investigation also gave additional scientific support to the use of C. chinensis to treat H. pylori-related gastrointestinal diseases in traditional Chinese medicine. Pal might be a potentially beneficial therapy for gastritis and peptic ulcers induced by H. pylori infection and other urease-related diseases.

Evaluation of Antifungal Effect of Silver Nanoparticles Against Microsporum canis, Trichophyton mentagrophytes and Microsporum gypseum.

Dermatophytosis is the common cutaneous infections in humans and animals, which is caused by the keratinophylic fungus called dermatophytes. In recent years, drugs resistance in pathogenic fungi, including dermatophyte strains to the current antifungals have been increased. The aim of this study was to evaluate the antifungal efficacy of AgNPs against Microsporum canis, Trichophyton mentagrophytes , and Microsporum gypseum. The antifungal susceptibility of nanosilver particles compared with griseofulvin (GR). Its efficacy was investigated against three strains of dermatophytes by both agar dilution and broth microdilution test (BMD). The average minimum inhibitory concentration (MIC) AgNPs on M. canis, T. mentagrophytes and M. gypseum were 200, 180 and 170 μg.mL-1, respectively. Whereas these strains showed MIC of 25, 100 and 50 μg.mL-1 for GR. Our finding indicated that the AgNPs was less active than GR but it had anti-dermatophytic effect.

A Portable 3D Printer System for the Diagnosis and Treatment of Multidrug-Resistant Bacteria

Summary Multidrug-resistant bacteria are a major threat to human health, but broad-spectrum antibiotics are losing efficacy. There is a need to screen a given drug against a bacterial infection outside of the laboratory. To address this need, we have designed and built an inexpensive and easy-to-use 3D-printer-based system that allows easily readable quantitative tests for the performance of antibacterial drugs. The platform creates a sterile diagnostic device by using 3D printing, and the device is filled automatically with growth medium, and then antibiotics are sprayed onto the medium by ink-jet technology. The sample for testing can be introduced via a fluid port, and the printer hot bed is used to incubate the device, allowing operation in the field. Combining advantages from various established tests of antibacterial performance, this allows the comparison of a specific antibiotics and bacteria. Also, this system can create and test several antibiotic formulations for therapeutic use, and we demonstrate this potential by investigating a mixture of pathogens that are only killed by a mixture of drugs.

Correlation of mupirocin resistance with biofilm production in methicillin-resistant Staphylococcus aureus from surgical site infections in a tertiary centre, Egypt.

The aim of this study was to detect mupirocin-resistant isolates from pus/wound swabs taken postoperatively in a tertiary centre in Egypt and to determine their ability to form biofilm in order to establish its correlation with mupirocin resistance. This was a prospective study including 513pus/wound swabs from patients suffering from postoperative surgical site infections over the period July 2013-January 2015. Samples were cultured and isolates were identified by coagulase activity, DNase test, mannitol fermentation by mannitol salt agar followed by API Staph 32. Oxacillin agar screen test, agar dilution test for mupirocin, and mupA gene detection by PCR were performed for all methicillin-resistant Staphylococcus aureus (MRSA) isolates. Biofilm detection was carried out by the microtitre plate and Congo red agar methods. Of the 161 S. aureus isolates identified, 73 (45.3%) were MRSA, among which 82.2% were mupirocin-susceptible and 17.8% were mupirocin-resistant. Among the resistant isolates, 38.5% showed low-level resistance and 61.5% were high-level mupirocin-resistant. The mupA gene was detected in 75.0% of high-level mupirocin-resistant strains and in none of the low-level mupirocin-resistant strains. Among the mupirocin-susceptible isolates, 95.0% were biofilm-producers and 5.0% did not produce biofilm. All mupirocin-resistant isolates produced biofilm. Moreover, 15.3% of high-level mupirocin-resistant strains were negative for the mupA gene but showed evidence of biofilm formation. In conclusion, biofilm formation may be suggested to play a role in mupirocin resistance besides the presence of a genetic element encoding abnormal isoleucyl-tRNA synthetase, however further studies are needed to confirm these findings.

An investigation of the bactericidal activity of chlorhexidine digluconateagainst multidrug-resistant hospital isolates.

Hospital infections are among the most prominent medical problems around the world. Using proper biocidesin an appropriate way is critically important in overcoming this problem. Several reports have suggested that microorganisms may develop resistance or reduce their susceptibility to biocides, similar to the case with antibiotics. In this study we aimed to determine the antimicrobial activity of chlorhexidine digluconate against clinical isolates. The susceptibility of 120 hospital isolated strains of 7 bacterial genera against chlorhexidine digluconate was determined by agar dilution test, using minimum inhibitory concentration (MIC) values and the EN 1040 Basic Bactericidal Activity Test to determine the bactericidal activity. According to MIC values,Pseudomonas aeruginosaandStenotrophomonas maltophiliawere found to be less susceptible to chlorhexidine digluconate. Quantitative suspension test results showed that4% chlorhexidine digluconatewas effective against antibiotic resistant and susceptible bacteria after 5 min of contact time and can be safelyused inour hospital. However, concentrations below 4% chlorhexidine digluconate caused a decrease in bactericidal activity, especially for Staphylococcus aureus and P. aeruginosa. It is crucial to use biocides at appropriate concentrations and to performsurveillancestudies to trace resistanceor low susceptibility patterns ofS. aureus, P. aeruginosa,and other hospital isolates.

The Comparative Evaluation of the Antimicrobial Effect of Propolis with Chlorhexidine against Oral Pathogens: An In Vitro Study

This study aimed to compare the antimicrobial effectiveness of ethanolic extract of propolis (EEP) to chlorhexidine gluconate (CHX) on planktonic Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Lactobacillus salivarius subsp. salivarius, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Staphylococcus aureus, Enterococcus faecalis, Actinomyces israelii, Candida albicans, and their single-species biofilms by agar dilution and broth microdilution test methods. Both agents inhibited the growth of all planktonic species. On the other hand, CHX exhibited lower minimum bactericidal concentrations than EEP against biofilms of A. actinomycetemcomitans, S. aureus, and E. faecalis whereas EEP yielded a better result against Lactobacilli and P. intermedia. The bactericidal and fungicidal concentrations of both agents were found to be equal against biofilms of Streptecocci, P. gingivalis, A. israelii, and C. albicans. The results of this study revealed that propolis was more effective in inhibiting Gram-positive bacteria than the Gram-negative bacteria in their planktonic state and it was suggested that EEP could be as effective as CHX on oral microorganisms in their biofilm state.

Antimicrobial Effect of Malpighia Punicifolia and Extension of Water Buffalo Steak Shelf-Life.

In the present study, a multiple approach was used to characterize Malpighia punicifolia extract and to evaluate its inhibitory activity against several meat spoilage bacteria. First, volatile fraction, vitamins and phenolic compounds of the extract obtained by supercritical fluid extraction were determined by GC‐MS and HPLC. Then, the antimicrobial action of the extract was in vitro evaluated against Pseudomonas putida DSMZ 291T, Pseudomonas fluorescens DSMZ 50009T, Pseudomonas fragi DSMZ 3456T, and Brochothrix thermosphacta DSMZ 20171T by the agar well diffusion assay and by the agar dilution test. Based on the results of the minimum inhibitory concentration (MIC) against the assayed bacteria, 4 different concentrations of the extract were used in a challenge test on water buffalo steaks stored for 21 d at 4 °C. Results of chemical analyses showed that M. punicifolia extract is characterized by the presence of several compounds, already described for their antimicrobial (phenolic acids, flavonones, and furanes) and antioxidant (ascorbic acid) properties. The in vitro detection of antimicrobial activities highlighted that the extract, used at 8% concentration, was able to inhibit all the target bacteria. Moreover, very low MIC values (up to 0.025%) were detected. In situ tests, performed on water buffalo steaks treated with the extract in the concentration range 0.025% to 0.05%, showed a strong inhibition of both intentionally inoculated bacteria and naturally occurring microorganisms. Positive results, in terms of color and odor, were also observed during the entire storage of steaks preserved with the extract.

Analysis of the transcriptome in a multi-drug resistant uropathogenic Escherichia coli strain treated with water decoction of rhizoma coptidis by using RNA-seq

Objective To investigate the molecular mechanism of the inhibitory effects of rhizoma coptidis on multi-drug resistant uropathogenic Escherichia coli. Methods High-throughput RNA sequencing (RNA-seq) was performed to investigate the transcriptome in a multi-drug resistant uropathogenic Escherichia coli strain (NB8) treated with water decoction of rhizoma coptidis. Agar dilution test was used to determine the minimal inhibitory concentration (MIC) of water decoction of rhizoma coptidis against the NB8 strain. A growth curve was drawn to evaluate the effects of water decoction of rhizoma coptidis on the growth of NB8 strain. Total RNAs were extracted from the NB8 strain after treated with the water decoction of rhizoma coptidis for 30 minutes and then synthetized to cDNA by reverse transcription after screening out the rRNAs. The HiSeq 2000 sequencing system was used for transcriptome sequencing. The TopHat software was used to map and analyze the RNA-Seq reads, and then Cufflinks was run to assemble transcripts and estimate their abundances. The differential expression, GO enrichment and KEGG metabolic pathway were further analyzed. The NB8 strain dealt with normal saline was used as negative control. Results The MIC of water decoction of rhizoma coptidis to NB8 strain was 12.5 mg/ml. There were 3665 genes expressed in NB8 strain treated with water decoction of rhizoma coptidis and 3430 genes expressed in NB8 strain treated with normal saline. The number of differentially expressed genes was 1428 including 921 up-regulated genes and 507 down-regulated genes. Those differentially expressed genes mainly enriched in the modules of binding and catalysis. The genes concerning cell adhesion, apoptosis and multicellular process were up-regulated, while those concerning the regulation of enzyme activities were down-regulated. Results of the KEGG metabolic pathway enrichment analysis showed that the genes concerning synthetic pathway of LPS were significantly up-regulated as well as those encoding the repair polymerase Ⅲ that was involved in DNA replication. However, the genes concerning fatty acid metabolism, histidine metabolism, thiamine metabolism, folate metabolism and iron carrier in ribosome synthesis showed overall down-regulation. Conclusion The transcriptome in uropathogenic Escherichia coli strain treated with rhizoma coptidis was profiled. The main molecular mechanism of the inhibitory effects of rhizoma coptidis on uropathogenic Escherichia coli was to destroy the cell wall of Escherichia coli, affect the replication of DNA and regulate the transcription and translation of proteins. This study illustrated that the inhibitory effects of rhizoma coptidis on uropathogenic Escherichia coli were achieved in multiple levels. Key words: Escherichia coli; Transcriptome; Water decoction of rhizoma coptidis; RNA-seq; Differentially expressed genes; Functional enrichment

Determination of Disk Diffusion and MIC Quality Control Guidelines for Solithromycin, a Novel Fluoroketolide Antibacterial, against Neisseria gonorrhoeae.

This solithromycin quality control study was performed to establish quality control (QC) ranges for the N. gonorrhoeae ATCC 49226 control strain for MIC agar dilution testing (AD) and zones by disk diffusion testing (DD). The following ranges were established: AD, 0.03 to 0.25 μg/ml, and DD, 33 to 43 mm. In January 2015, the CLSI Subcommittee on Antimicrobial Susceptibility Testing approved these ranges, which will be important when evaluating solithromycin against clinical isolates of N. gonorrhoeae.