MICs Of Gentamicin Research Articles

Chemical characterization, antibacterial and antifungal activity of honey pots and pollen pots obtained from the stingless bee Tetragonisca angustula (Latreille, 1811).

This study aimed to determine the chemical composition of the ethanolic extracts of honey pots (EEHPTa) and pollen pots (EEPPTa) from the stingless bee Tetragonisca angustula, as well as to evaluate their antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and Candida tropicalis. The chemical composition was determined using HPLC-Q-TOF-MS/MS and HPLC-DAD, while antimicrobial assays were performed using the broth microdilution method. Eleven compounds were identified in EEHPTa and nine in EEPPTa, including cirsimaritin, 3'-prenylnaringenin, xanthohumol, lespedezaflavanone B, and 19α-hydroxyursolic acid. The extracts enhanced the action of norfloxacin against S. aureus (MIC reduced from 256 to 128μg/mL). With gentamicin, EEHPTa reduced the MIC from 17.95 to 11.31μg/mL, while EEPPTa reduced it to 12.69μg/mL. Only EEHPTa was effective with ampicillin, reducing the MIC from 71.83 to 31.7μg/mL. Against P. aeruginosa, EEHPTa decreased the MIC of gentamicin from 8 to 4μg/mL, whereas EEPPTa exhibited an antagonistic effect. For E. coli, only EEPPTa reduced the MIC from 64 to 57.01μg/mL. EEHPTa exhibited lower IC50 values against yeast (C. albicans: 65.47μg/mL; C. tropicalis: 29.79μg/mL) compared to EEPPTa (C. albicans: 820.7μg/mL; C. tropicalis: 809.9μg/mL). Both extracts enhanced the effect of fluconazole against C. albicans but showed no effect on the C. tropicalis strain. These findings highlight the therapeutic potential of the secondary metabolites in these extracts, reinforcing their importance in the development of new antimicrobial and antifungal strategies.

New Bacteriophage Pseudomonas Phage Ka2 from a Tributary Stream of Lake Baikal

Pseudomonas aeruginosa, an opportunistic pathogen, causes various biofilm-associated infections like pneumonia, infections in cystic fibrosis patients, and urinary tract and burn infections with high morbidity and mortality, as well as low treatment efficacy due to the extremely wide spread of isolates with multidrug resistance. Here, we report the new bacteriophage Pseudomonas phage Ka2 isolated from a tributary stream of Lake Baikal and belonging to the Pbunavirus genus. Transmission electron microscopy resolved that Pseudomonas phage Ka2 has a capsid of 57 ± 9 nm and a contractile and inflexible tail of 115 ± 10 nm in the non-contracted state. The genome consists of 66,310 bp with a GC content of 55% and contains 96 coding sequences. Among them, 52 encode proteins have known functions, and none of them are potentially associated with lysogeny. The bacteriophage lyses 21 of 30 P. aeruginosa clinical isolates and decreases the MIC of amikacin, gentamicin, and cefepime up to 16-fold and the MIC of colistin up to 32-fold. When treating the biofilms with Ka2, the biomass was reduced by twice, and up to a 32-fold decrease in the antibiotics MBC against biofilm-embedded cells was achieved by the combination of Ka2 with cefepime for the PAO1 strain, along with a decrease of up to 16-fold with either amikacin or colistin for clinical isolates. Taken together, these data characterize the new Pseudomonas phage Ka2 as a promising tool for the combined treatment of infections associated with P. aeruginosa biofilms.

Molecular Docking and Antibacterial Activity of Campesterol Derivatives Against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa Multiresistant Strains.

This work describes the evaluation the potentiating activity of antibiotics by campesterol (1) and its derivatives (2-11) against multiresistant strains of Staphylococcus aureus 10, Escherichia coli 06 and Pseudomonas aeruginosa 24 employing the microdilution test. When subjected to the in vitro potentiating activity bioassay, all compounds showed a potentiating effect associated with norfloxacin against E. coli and P. aeruginosa with a reduction in the MIC of the antibiotic of up to 75 %. These compounds also reduced the MIC of gentamicin by 37 % to 87 % in S. aureus and E. coli. Additionally, molecular docking studies were conducted to gain a deeper understanding of the interactions between the appropriate proteins and the most effective compounds (2, 4, 9, and 10 against E. coli; 1, 2, 3, 5, 8, and 9 against S. aureus), including antibiotics. This paper registers for the first time the in vitro and in silico studies on the action of compounds 1-11 in antibiotic potentiation.

Methyl gallate attenuates virulence and decreases antibiotic resistance in extensively drug-resistant Pseudomonasaeruginosa

Pseudomonas aeruginosa infections have become a serious threat to public health due to the increasing emergence of extensively antibiotic-resistant strains and high mortality rates. Therefore, the search for new therapeutic alternatives has become crucial. In this study, the antivirulence and antibacterial activity of methyl gallate was evaluated against six clinical isolates of extensively antibiotic-resistant P. aeruginosa. Methyl gallate exhibited minimal inhibitory concentrations of 256-384μg/mL; moreover, the use of subinhibitory concentrations of the compound inhibited biofilm formation, swimming, swarming, proteolytic activity, and pyocyanin production. Methyl gallate plus antipseudomonal antibiotics showed a synergistic effect by reduced the MICs of ceftazidime, gentamicin and meropenem. Furthermore, the potential therapeutic effect of methyl gallate was demonstrated in an infection model. This study evidenced the antivirulence and antimicrobial activity of methyl gallate as a therapeutic alternative against P. aeruginosa.

Ceftazidime/avibactam resistance is associated with PER-3-producing ST309 lineage in Chilean clinical isolates of non-carbapenemase producing Pseudomonas aeruginosa.

Ceftazidime/avibactam (CZA) is indicated against multidrug-resistant Pseudomonas aeruginosa, particularly those that are carbapenem resistant. CZA resistance in P. aeruginosa producing PER, a class A extended-spectrum β-lactamase, has been well documented in vitro. However, data regarding clinical isolates are scarce. Our aim was to analyze the contribution of PER to CZA resistance in non-carbapenemase-producing P. aeruginosa clinical isolates that were ceftazidime and/or carbapenem non-susceptible. Antimicrobial susceptibility was determined through agar dilution and broth microdilution, while bla PER gene was screened through PCR. All PER-positive isolates and five PER-negative isolates were analyzed through Whole Genome Sequencing. The mutational resistome associated to CZA resistance was determined through sequence analysis of genes coding for PBPs 1b, 3 and 4, MexAB-OprM regulators MexZ, MexR, NalC and NalD, AmpC regulators AmpD and AmpR, and OprD porin. Loss of bla PER-3 gene was induced in a PER-positive isolate by successive passages at 43°C without antibiotics. Twenty-six of 287 isolates studied (9.1%) were CZA-resistant. Thirteen of 26 CZA-resistant isolates (50%) carried bla PER. One isolate carried bla PER but was CZA-susceptible. PER-producing isolates had significantly higher MICs for CZA, amikacin, gentamicin, ceftazidime, meropenem and ciprofloxacin than non-PER-producing isolates. All PER-producing isolates were ST309 and their bla PER-3 gene was associated to ISCR1, an insertion sequence known to mobilize adjacent DNA. PER-negative isolates were classified as ST41, ST235 (two isolates), ST395 and ST253. PER-negative isolates carried genes for narrow-spectrum β-lactamases and the mutational resistome showed that all isolates had one major alteration in at least one of the genes analyzed. Loss of bla PER-3 gene restored susceptibility to CZA, ceftolozane/tazobactam and other β-lactamsin the in vitro evolved isolate. PER-3-producing ST309 P. aeruginosa is a successful multidrug-resistant clone with blaPER-3 gene implicated in resistance to CZA and other β-lactams.

Emergence of High-Level Gentamicin Resistance in Streptococcus agalactiae Hypervirulent Serotype IV ST1010 (CC452) Strains by Acquisition of a Novel Integrative and Conjugative Element.

Streptococcus agalactiae (group B streptococci, GBS) is responsible for severe infections in both neonates and adults. Currently, empiric antimicrobial therapy for sepsis and meningitis is the combined use of penicillin and gentamicin due to the enhanced bactericidal activity. However, high-level gentamicin resistance (HLGR) abrogates the synergism. The rate of HLGR was investigated within a dataset of 433 GBS strains collected from cases of invasive disease in both adults and neonates as well as from pregnant carriers. GBS isolates (n = 20, 4.6%) presented with HLGR (gentamicin MIC breakpoint >1024 mg/L) that was differently diffused between strains from adults or neonates (5.2% vs. 2.8%). Notably, 70% of HLGR GBS strains (14 isolates) were serotype IV. Serotype IV HLGR-GBS isolates were susceptible to all antibiotics tested, exhibited the alpha-C/HvgA/PI-2b virulence string, and belonged to sequence type 1010 (clonal complex (CC) 452). The mobile element that harbored the HLGR aac(6')-aph(2)″ gene is a novel integrative and conjugative element (ICE) about 45 kb long, derived from GBS 515 ICE tRNALys. The clonal expansion of this HLGR hypervirulent serotype IV GBS CC452 sublineage may pose a threat to the management of infections caused by this strain type.

2809. In Vitro Synergy of Antibiotic Combinations Against Enterococcus gallinarum

Abstract Background Enterococci are an important cause of endocarditis, particularly involving prosthetic valves. The prevalence of E. gallinarum has been increasing in recent years. Unlike E. faecalis, no information is available regarding the bactericidal activity of ampicillin alone or combined with gentamicin or ceftriaxone against E. gallinarum. Methods Ten blood culture isolates of E. gallinarum were studied. The isolates came from unique patients at a single hospital. MICs of ampicillin, ceftriaxone and gentamicin were done by the CLSI microdilution method. ATCC strains E. faecalis 29212 and P. aeruginosa 27853 were used as controls. Time-kill studies were performed in CA-MHB for ampicillin 10 mg/L, ceftriaxone 10 mg/L, gentamicin at 0.25 x MIC up to a maximum of 4 mg/L, ampicillin + ceftriaxone, and ampicillin + gentamicin. Bactericidal activity was defined as a 1,000-fold decrease in the log10 cfu/mL at 24 h. Synergy was defined as a 100-fold decrease in the log10 cfu/ml at 24 h in a combination compared to each single agent. Results The MICs of ampicillin ranged from 1 to 8 mg/L, gentamicin from 2 to 16 mg/L, and ceftriaxone were all ≥ 32 mg/L. Results of the time-kill studies are summarized in the Table. Ampicillin alone was bactericidal against a single isolate. For that isolate, testing of ampicillin at 0.25 x MIC with gentamicin demonstrated synergy. The combination of ampicillin + ceftriaxone was synergistic against a single isolate, but was otherwise similar to ampicillin alone. Ampicillin + gentamicin was bactericidal against all isolates and was synergistic against all but one isolate. Conclusion Ampicillin alone was generally not bactericidal against E. gallinarum but had synergistic bactericidal activity when combined with gentamicin, similar to the findings with E. faecalis. However, unlike E. faecalis, ampicillin + ceftriaxone was not bactericidal against most isolates of E. gallinarum. These findings support the suggestion that ampicillin + gentamicin might be the optimal treatment for endovascular infections caused by E. gallinarum. Disclosures All Authors: No reported disclosures

Trends of feline Escherichia coli minimum inhibitory concentrations over 14 years illustrate the need for judicious antimicrobial use in cats.

This study aims to assess the antimicrobial resistance (AMR) trends among Escherichia coli isolated from cats between 2008 and 2022, utilizing MIC data, within a one-health framework. The study analyzed MIC results from 1,477 feline E coli isolates that were obtained from samples submitted to the Cornell University Animal Health Diagnostic Center, primarily from the northeastern US. MIC values were categorized as susceptible or not susceptible using the Clinical and Laboratory Standards Institute breakpoints. Multidrug resistance (MDR) was analyzed using a Poisson regression model. Additionally, accelerated failure time models were employed to analyze MIC values. Out of the 1,477 E coli isolates examined, 739 (50%) showed susceptibility to all tested antimicrobials. Among the tested antimicrobials, cefazolin (69%) and ampicillin (74% for urinary tract isolates) exhibited the lowest susceptibility. Overall, 15% of isolates were not susceptible to cefovecin. E coli isolates were highly susceptible (> 95%) to antibiotics typically reserved for human use. Almost one-third of the isolates were classified as MDR, with nonurinary isolates more likely to exhibit an MDR pattern. A decrease in MICs for fluoroquinolones and gentamicin in recent years was identified. However, MICs for cephalexin increased from 2016 to 2022 and cefovecin from 2012 to 2019. This study highlights the challenge of AMR in feline medicine, emphasizing the importance of responsible antimicrobial use and surveillance to address E coli AMR. The related Currents in One Health by Cazer et al, JAVMA, December 2023, addresses additional feline antimicrobial stewardship topics.

Toward a Method for Harmonized Susceptibility Testing of Mycoplasma bovis by Broth Microdilution.

Mycoplasma bovis is a fastidious pathogen of cattle causing massive economic losses in the calf and dairy industries worldwide. Since there is no approved standard method for antimicrobial susceptibility testing (AST) of M. bovis, the Clinical and Laboratory Standards Institute has requested the development of a suitable method. Therefore, this study aimed at developing a method for harmonized broth microdilution AST of M. bovis. For this, 131 M. bovis field isolates and M. bovis strain DSM 22781T were collected and macrorestriction analysis was performed to select 15 epidemiologically unrelated M. bovis strains for method validation steps. To select a suitable broth for AST of M. bovis, growth determinations were performed using five media and growth curves were compiled. Then, susceptibility testing was performed considering the exact (precondition of five identical MICs) and essential (MIC mode, accepting a deviation of ±1 dilution step) MIC agreements to evaluate the reproducibility of MIC values using a panel of 16 antimicrobial agents. Subsequently, the remaining field isolates were tested and the suitability of quality control (QC) strains was assessed. Growth experiments showed that SP4 broth was the only one of the five media that yielded sufficient growth of M. bovis. Therefore, it was selected as the test medium for AST and homogeneous MIC values were obtained (exact and essential agreements of 36 to 100% and 92 to 100%, respectively). For all other isolates tested, easy-to-read MIC endpoints were determined with this medium. High overall MIC50 and/or MIC90 values were observed for aminoglycosides and macrolides, and some isolates showed elevated MICs of fluoroquinolones, gentamicin, and/or tiamulin. Since the MICs of four commonly used QC strains were partially not within their ranges, a 20-fold MIC testing of M. bovis DSM 22781T was performed and met the criteria for a new QC strain. For harmonized AST of M. bovis, SP4 broth seems to be suitable with an incubation time of 72 ± 2 h and further validation of M. bovis DSM 22781T as a future QC strain is recommended.

Tigecycline Resistance-Associated Mutations in the MepA Efflux Pump in Staphylococcus aureus.

Tigecycline is an important antibacterial drug for treating infection by clinical multidrug-resistant bacteria, and tigecycline-resistant Staphylococcus aureus (TRSA) has been increasingly reported in recent years. Notably, only rpsJ and mepA are associated with the tigecycline resistance of S. aureus. The mepA gene encodes MepA efflux pumps, and the overexpression of mepA has been confirmed to be directly related to tigecycline resistance. Although the mutations of MepA widely occur, the associations between TRSA and mutations of MepA are still unclear. In this study, we explored mutations in the mepA genes from various sources. Then, tigecycline resistance-associated mutations T29I, E287G, and T29I+E287G in MepA were identified, and their effects were evaluated through mutant deletion and complementation, tigecycline accumulation assay, and molecular docking experiments. Results showed that the MICs of tigecycline, gentamicin, and amikacin increased in special complementary transformants and recovered after the addition of the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The tigecycline accumulation assay of the mepA-deleted mutant strain and its complementary transformants showed that T29I, E287G, and T29I+E287G mutations promoted tigecycline efflux, and molecular docking showed that mutations T29I, E287G, and T29I+E287G decreased the binding energy and contributed to ligand binding. Moreover, we inferred the evolutionary trajectory of S. aureus under the selective pressure of tigecycline in vitro. Overall, our study indicated that mutations in MepA play important roles in tigecycline resistance in S. aureus. IMPORTANCE Previous analysis has shown that overexpression of MepA is an exact mechanism involved in tigecycline resistance apart from the rpsJ mutation and is usually dependent on the mutant mepR. However, no research has evaluated the effects of diverse mutations discovered in TRSA in MepA. This study demonstrates that the mutations in MepA confer resistance to tigecycline without overexpression and provides genotypic references for identifying TRSA. Although tigecycline resistance-associated mutations in MepA identified in this study have not been observed in clinical isolates, the mechanism should be explored given that S. aureus strains are prevalent in the environment. Measures should be implemented to contain TRSA within the time window before tigecycline resistance-associated mutations in MepA are prevalent.

Carbonic Anhydrase Inhibition as a Target for Antibiotic Synergy in Enterococci.

Enterococcus faecalis is a hospital-associated opportunistic pathogen that can cause infections with high mortality, such as infective endocarditis. With an increasing occurrence of multidrug-resistant enterococci, there is a need for alternative strategies to treat enterococcal infections. We isolated a gentamicin-hypersusceptible E. faecalis strain from a patient with infective endocarditis that carried a mutation in the alpha-carbonic anhydrase (α-CA) and investigated how disruption of α-CA sensitized E. faecalis to killing with gentamicin. The gentamicin-hypersusceptible α-CA mutant strain showed increased intracellular gentamicin uptake in comparison to an isogenic strain encoding full-length, wild-type α-CA. We hypothesized that increased gentamicin uptake could be due to increased proton motive force (PMF), increased membrane permeability, or both. We observed increased intracellular ATP production in the α-CA mutant strain, suggesting increased PMF-driven gentamicin uptake contributed to the strain's gentamicin susceptibility. We also analyzed the membrane permeability and fatty acid composition of isogenic wild-type and α-CA mutant strains and found that the mutant displayed a membrane composition that was consistent with increased membrane permeability. Finally, we observed that exposure to the FDA-approved α-CA inhibitor acetazolamide lowered the gentamicin MIC of eight genetically diverse E. faecalis strains with intact α-CA but did not change the MIC of the α-CA mutant strain. These results suggest that α-CA mutation or inhibition increases PMF and alters membrane permeability, leading to increased uptake of gentamicin into E. faecalis. This connection could be exploited clinically to provide new combination therapies for patients with enterococcal infections. IMPORTANCE Enterococcal infections can be difficult to treat, and new therapeutic approaches are needed. In studying an E. faecalis clinical strain from an infected patient, we found that the bacteria were rendered hypersusceptible to aminoglycoside antibiotics through a mutation that disrupted the α-CA. Our follow-on work suggested two different ways that α-CA disruption causes increased gentamicin accumulation in E. faecalis: increased proton motive force-powered uptake and increased membrane permeability. We also found that a mammalian CA inhibitor could sensitize a variety of E. faecalis strains to killing with gentamicin. Given that mammalian CA inhibitors are frequently used to treat conditions such as glaucoma, hypertension, and epilepsy, our findings suggest that these "off-the-shelf" inhibitors could also be useful partner antibiotics for the treatment of E. faecalis infections.

High-level in vitro resistance to gentamicin acquired in a stepwise manner in Neisseria gonorrhoeae.

Gentamicin is used in several alternative treatments for gonorrhoea. Verified clinical Neisseria gonorrhoeae isolates with gentamicin resistance are mainly lacking and understanding the mechanisms for gonococcal gentamicin resistance is imperative. We selected gentamicin resistance in gonococci in vitro, identified the novel gentamicin-resistance mutations, and examined the biofitness of a high-level gentamicin-resistant mutant. Low- and high-level gentamicin resistance was selected in WHO X (gentamicin MIC = 4 mg/L) on gentamicin-gradient agar plates. Selected mutants were whole-genome sequenced. Potential gentamicin-resistance fusA mutations were transformed into WT strains to verify their impact on gentamicin MICs. The biofitness of high-level gentamicin-resistant mutants was examined using a competitive assay in a hollow-fibre infection model. WHO X mutants with gentamicin MICs of up to 128 mg/L were selected. Primarily selected fusA mutations were further investigated, and fusAR635L and fusAM520I + R635L were particularly interesting. Different mutations in fusA and ubiM were found in low-level gentamicin-resistant mutants, while fusAM520I was associated with high-level gentamicin resistance. Protein structure predictions showed that fusAM520I is located in domain IV of the elongation factor-G (EF-G). The high-level gentamicin-resistant WHO X mutant was outcompeted by the gentamicin-susceptible WHO X parental strain, suggesting lower biofitness. We describe the first high-level gentamicin-resistant gonococcal isolate (MIC = 128 mg/L), which was selected in vitro through experimental evolution. The most substantial increases of the gentamicin MICs were caused by mutations in fusA (G1560A and G1904T encoding EF-G M520I and R635L, respectively) and ubiM (D186N). The high-level gentamicin-resistant N. gonorrhoeae mutant showed impaired biofitness.

Effects of Sub-Minimum Inhibitory Concentrations of Gentamicin on Alginate Produced by Clinical Isolates of Pseudomonas aeruginosa.

Bacterial virulence factors may be influenced by sub-minimum inhibitory concentrations (sub-MICs) of antibiotics. The main purpose of this study was to investigate the effects of gentamicin at sub-MICs (0.5 MIC and 0.25 MIC) on alginate production of clinical isolates of Pseudomonas aeruginosa. The minimum inhibitory concentrations of gentamicin against 88 clinical isolates of P. aeruginosa were determined using the broth microdilution method. Alginate production of the isolates in the absence and presence of gentamicin at sub-MICs was assessed by the carbazole method. The presence of alginate in clinical isolates was confirmed by the detection of alginate genes (algD and algU) using the PCR method. All the isolates had the ability of alginate production and were positive for algD and algU genes. sub-MICs of gentamicin significantly increased alginate production of 34 isolates (38.6%). On the other hand, in 49 isolates (55.7%), alginate production was significantly increased after treatment with sub-MICs of gentamicin. In five isolates (5.7%), the alginate production was reduced in exposure to 0.5 MIC of gentamicin while it was increased by gentamicin at 0.25 MIC. This study showed different effects of gentamicin at sub-MICs on the alginate production of clinical isolates of P. aeruginosa. Further research is highly recommended to understand the mechanism of different responses of P. aeruginosa isolates to the exposure of sub-MICs of gentamicin.

Optimization of antibacterial activity of essential oil mixture obtained from three medicinal plants: Evaluation of synergism with conventional antibiotics and nanoemulsion effectiveness

• Mixture design of L. maroccana, T. vulgaris and A. leucotricus were established. • Synergistic interactions were recorded and optimized EO mixture was defined. • Optimal mixture decreased the antibiotics MICs by 2- to 64-fold. • Optimized EO mixture showed high antibacterial effect in nanoemulsion. The present study aimed to optimize the antibacterial activity of essential oil (EO) mixture obtained from Ammodaucus leucotrichus Cosson, Thymus vulgaris L. and Lavandula maroccana (Murb.) against three multi-drug resistant bacteria, using augmented simplex-centroid design. GC–MS analysis showed that A. leucotrichus EO was characterized by the abundance of l -perillaldehyde (50.39%), d -limonene (21.61%) and bornyl angelate (7.09%). T. vulgaris EO was dominated by thymol (42.34%), p -cymene (19.71%) and γ-terpinene (11.50%), while carvacrol (71.62%) represents the main component of L. maroccana EO. Regarding antibacterial assays, individual EOs displayed moderate activity with MIC and MBC values ranging from 0.06% to 5.00%, while the coefficient terms of the different tested combinations revealed synergistic interactions. The results of predicted optimization showed that the optimal EOs mixture composed by 55% of T. vulgaris, 41% of L. maroccana and 4% of A. leucotrichus, significantly decreased the MICs against Staphylococcus aureus (0.0225%), Escherichia coli (0.05%) and Pseudomonas aeruginosa (0.688%). The optimal mixture exhibited a significant decrease in the MICs of gentamicin (4-fold) and amoxicillin (2- to 64-fold), and achieve the highest antibacterial effect in nanoemulsion formulation. The optimized EO mixture based on the studied plants should be considered as a potential alternative to control foodborne, and resistant bacteria.

Gentamicin Variably Affects amrZ and rhl gene Expression in Swarmer Cells of Pseudomonas aeruginosa

Swarming is one of the most important virulence factors used by bacteria to invade new sites. This study aimed to test the effects of gentamicin on swarming motility of Pseudomonas aeruginosa, both phenotypically and molecularly. The present results revealed that 11/25 isolates had gentamicin MIC of 1024 µg/ml. However, gentamicin at sub-minimal inhibitory concentration significantly (P< 0.05) reduced the diameter of swarming in all P. aeruginosa isolates. Noticeably the mean and median swarming diameter before treatment with gentamicin 5.557 and 5.816 cm respectively had significantly (P < 0.001) reduced to 0.871 and 0.766 cm respectively. At the molecular level, amrZ (a global regulator of multiple genes) and rhl (responsible for rhamnolipid production) were variably affected by gentamicin. More likely it can be concluded that amrZ and rhl are not fully responsible for swarming in P. aeruginosa isolates.

Identifying patient-level risk factors associated with non-β-lactam resistance outcomes in invasive MRSA infections in the United States using chain graphs.

BackgroundMRSA is one of the most common causes of hospital- and community-acquired infections. MRSA is resistant to many antibiotics, including β-lactam antibiotics, fluoroquinolones, lincosamides, macrolides, aminoglycosides, tetracyclines and chloramphenicol.ObjectivesTo identify patient-level characteristics that may be associated with phenotype variations and that may help improve prescribing practice and antimicrobial stewardship.MethodsChain graphs for resistance phenotypes were learned from invasive MRSA surveillance data collected by the CDC as part of the Emerging Infections Program to identify patient level risk factors for individual resistance outcomes reported as MIC while accounting for the correlations among the resistance traits. These chain graphs are multilevel probabilistic graphical models (PGMs) that can be used to quantify and visualize the complex associations among multiple resistance outcomes and their explanatory variables.ResultsSome phenotypic resistances had low connectivity to other outcomes or predictors (e.g. tetracycline, vancomycin, doxycycline and rifampicin). Only levofloxacin susceptibility was associated with healthcare-associated infections. Blood culture was the most common predictor of MIC. Patients with positive blood culture had significantly increased MIC of chloramphenicol, erythromycin, gentamicin, lincomycin and mupirocin, and decreased daptomycin and rifampicin MICs. Some regional variations were also observed.ConclusionsThe differences in resistance phenotypes between patients with previous healthcare use or positive blood cultures, or from different states, may be useful to inform first-choice antibiotics to treat clinical MRSA cases. Additionally, we demonstrated multilevel PGMs are useful to quantify and visualize interactions among multiple resistance outcomes and their explanatory variables.

Chemical profile, antioxidant and antimicrobial effects of essential oil from the Moroccan endemic plant cladanthus scariosus (L.)

ABSTRACT In this study, chemical profile, antioxidant potential and antimicrobial interactions with conventional antimicrobials of essential oil (EO) isolated from Cladanthus scariosus aerial parts were investigated. The EO was analyzed by GC-MS (Gas Chromatography-Mass spectrometry). Forty-three components representing 98.7% of the oil were identified. The main components were o-cymene, caryophyllene oxide, γ-terpinene and α-sabinene. Furthermore, the antioxidant and antimicrobial activities against ten bacteria and two Candida strains were assessed. The results showed an interesting antioxidant effect with IC50 of 1.04 ± 0.04 mg/mL, 0.55 ± 0.01 mg/mL and 0.36 ± 0.14 mg/mL, respectively, for DPPH (2,2-Diphenyl-1-Picrylhydrazyl), FRAP (Ferric-Reducing-Ability-Power), and ABTS (2-2’azinobis-3-ethylbenzothiazoline-6-sulphonate) methods. The EO exhibited a strong antimicrobial activity, especially against Methicillin-resistant Staphylococcus aureus and Candida glabrata. In combination with antimicrobials, the studied EO revealed a high synergism effect in reducing gentamicin MICs of bacterial strains by 128- to 256-fold and by 2- to 8-fold Fluconazole MICs against Candida strains.

Propolis antibacterial and antioxidant synergisms with gentamicin and honey.

Assess the antibacterial and antioxidant effects of ethanol extracts of Portuguese propolis samples when combined with gentamicin, a widely used aminoglycoside antibiotic, or with honey, collected from the same apiary as propolis. Using the agar dilution method and DPPH free radical scavenging assay, antimicrobial and antioxidant synergistic effects between propolis and gentamicin or honey were assessed. Synergism between propolis and gentamicin was observed for all the tested extracts and against all the indicator bacteria, with particular interest against the Methicillin-Resistant Staphylococcus aureus (MRSA) with a threefold decrease of the gentamicin MIC if mixed with 25µgml-1 propolis. Likely to propolis and gentamicin, mixtures of sub-MIC concentrations of propolis and honey enhanced the antibacterial action of each individual natural product against the majority of the strains. However, propolis antioxidant capacity decreased along with higher honey content in the mixture. Propolis has a strong synergistic effect when combined with gentamicin, allowing the reduction of the therapeutic dose of this drug. Propolis and honey mixtures also display a stronger antibacterial effect than the activity exhibited by each sample when tested individually, whereas the high antioxidant capacity of propolis seems to be not affected when in combination with honey. When mixed with honey, propolis antioxidant potential is maintained, or just slightly reduced up to 1:1 dilution, and show synergistic antibacterial effects, allowing to optimize the use of this usually scarce natural resource. Also, considering the antibiotic resistance problem, natural beehive products, alone or in the mixture, are promising alternatives to retard the outbreak of microbial resistance.

Detection and characterisation of 16S rRNA methyltransferase-producing Pseudomonas aeruginosa from the UK and Republic of Ireland from 2003–2015

16S rRNA methyltransferase (16S RMTase) genes confer high-level aminoglycoside resistance, reducing treatment options for multidrug-resistant Gram-negative bacteria. Pseudomonas aeruginosa isolates (n=221) exhibiting high-level pan-aminoglycoside resistance (amikacin, gentamicin and tobramycin MICs ≥64, ≥32 and ≥32 mg/L, respectively) were screened for 16S RMTase genes to determine their occurrence among isolates submitted to a national reference laboratory from December 2003 to December 2015. 16S RMTase genes were identified using two multiplex PCRs, and whole-genome sequencing (WGS) was used to identify other antibiotic resistance genes, sequence types (STs) and the genetic environment of 16S RMTase genes. 16S RMTase genes were found in 8.6% (19/221) of isolates, with rmtB4 (47.4%; 9/19) being most common, followed by rmtD3 (21.1%; 4/19), rmtF2 (15.8%; 3/19) and single isolates harbouring rmtB1, rmtC and rmtD1. Carbapenemase genes were found in 89.5% (17/19) of 16S RMTase-positive isolates, with blaVIM (52.9%; 9/17) being most common. 16S RMTase genes were found in 'high-risk' clones known to harbour carbapenemase genes (ST233, ST277, ST357, ST654 and ST773). Analysis of the genetic environment of 16S RMTase genes identified that IS6100 was genetically linked to rmtB1; IS91 to rmtB4, rmtC or rmtD3; ISCR14 to rmtD1; and rmtF2 was linked to Tn3, IS91 or Tn1721. Although 16S RMTase genes explained only 8.6% of pan-aminoglycoside resistance in the P. aeruginosa isolates studied, the association of 16S RMTase genes with carbapenemase-producers and 'high-risk' clones highlights that continued surveillance is required to monitor spread as well as the importance of suppressing the emergence of dually-resistant clones in hospital settings.

Different effects of sub-minimum inhibitory concentrations of gentamicin on the expression of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa.

Background and Objectives:Antibiotics at sub-minimum inhibitory concentrations (sub-MIC) may alter bacterial virulence factors. The objective of this study was to investigate the effect of gentamicin at sub-MIC concentrations on the expression of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa.Materials and Methods:The broth microdilution method was used to determine the MIC of gentamicin for three P. aeruginosa clinical isolates (P1-P3) and standard strains (PAO1 and 8821M). Alginate production and biofilm formation of the bacteria in the presence and absence of sub-MIC concentrations of gentamicin were measured using microtiter plate and carbazole assay, respectively. The real-time PCR method was used to determine the effect of gentamicin at sub-MIC concentrations on the expression level of genes involved in biofilm formation (pelA and pslA) and alginate production (algD and algU).Results:Gentamicin at sub-MIC concentrations significantly reduced alginate production, biofilm formation, and the expression of alginate and biofilm-encoding genes in clinical isolate P1. This inhibitory effect was also observed on the alginate production of 8821M strain and biofilm formation of PAO1strain. In clinical isolates, P2 and P3, alginate production, biofilm formation, and the expression of alginate and biofilm-encoding genes were significantly increased in exposure to sub-MIC concentrations of gentamicin.Conclusion:This study showed that different phenotypic changes in clinical isolates and standard strains of P. aeruginosa in exposure to sub-MIC concentrations of gentamicin are associated with changes in the expression of virulence genes. Further researches are required to understand the mechanisms involved in regulating the expression of virulence genes after exposure to sub-MIC concentrations of antibiotics.