Aminoglycosides Gentamicin Research Articles

Development of a dual-antimicrobial counterselection method for markerless genetic engineering of bacterial genomes.

Markerless genetic engineering of bacterial genomes is commonly performed by two-step homologous recombination methods using vectors carrying flanking regions of the target gene for site-specific vector integration and counterselection markers to provide positive selection pressure on the second recombination step resulting in vector excision. Here, we provide the proof-of-principle of a novel counterselection method that exploits antagonistic activities between bactericidal and bacteriostatic antibiotics and which can provide selection pressure on the second recombination step by selective killing of bacteria retaining the antibiotic selection marker. This method was optimized for the bacterial pathogens Listeria monocytogenes and Neisseria meningitidis by screening for antagonistic activities between the bactericidal aminoglycosides kanamycin, streptomycin, and gentamicin in combination with the bacteriostatic antibiotics chloramphenicol and erythromycin. The largest difference in selective killing of both L. monocytogenes and N. meningitidis containing an antibiotic selection marker versus wild-type bacteria was observed for the combination of erythromycin, gentamicin, and ermC as antibiotic selection marker. Therefore, this combination was used to generate two markerless deletion mutants for both L. monocytogenes and N. meningitidis. After applying the dual-antimicrobial selection pressure on cultures during the second recombination step, surviving colonies were replica plated on agar with and without erythromycin. On average, 12-13% of the randomly selected bacterial colonies had lost the selection marker due to a second recombination event and approximately half of these colonies were the desired markerless in-frame deletion mutants. Therefore, this method proved to be easy and fast and should be applicable to a wide variety of bacterial species.

Aminoglycoside Resistance Genes in Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen associated with serious hospital acquired infections. It is resistant to multiple antibiotics. The therapy for infections due to this bacterium is a combination of aminoglycosides with carbapenem antibiotics. There are recent reports of the prevalence of ami-noglycoside resistance among A. baumannii. The aim of the present study was to determine the prevalence of phosphotransferases APH (3')-Via (aphA6), acetyltransferases AAC (3)-Ia (aacC1), nucleotidyl transferases ANT (2'')-Ia (aadB), and ANT (3") -Ia (aadA1) genes among clinical isolates of A. baumannii and its relation to resis-tance to amikacin and gentamicin. The study included all clinical samples from intensive care units (ICUs) patients with suspected hospital acquired infections. Positive cultures were identified by Gram stain and complete biochemical identifications. An-tibiotic susceptibility was carried out by disc diffusion method. Minimum inhibitory concentration determination (MIC) to amikacin and gentamicin was performed by microdilution method. Polymerase chain reaction (PCR) was performed for detection of aadB, aadA1, aphA6, aadC1 genes. The study included 1,200 bacterial isolates from patients admitted to ICUs during the period of the study. A. baumannii represented 100 isolates from Gram negative bacilli. The study of MIC of A. baumannii to amikacin and gentamicin revealed that 50 (50%) of the isolates had resistance by MIC study with 36 (72%) of those having high level aminoglycoside resistance (HLAR) and 14 (28%) had non-high-level aminoglycoside resistance. The most common prevalent resistant genes among A. baumannii resistance to aminoglycosides was aadB (42%), fol-lowed by aphA6 (26%). Less prevalent genes were aadA1 (18%) and aacC1 (12%). There were 24 isolates with negative PCR for the studied genes. In a comparison between the prevalence of resistant genes among A. baumannii with HLAR and non HLAR, there was a non-significant increase of aphA6 (30.6%) and aadB (44.4%) in HLAR isolates compared to non HLAR (14.3%, 35.7%, respectively; p = 0.2), with a significant increase in aacC1 28.6% in HLAR compared to 5.6% in non HLAR (p = 0.02). Half of the clinical isolates of A. baumannii have resistance to the aminoglycosides amikacin and gen-tamicin. Most isolates have a high resistance level to aminoglycosides. The isolates have different types of amino-glycoside modifying genes. Further studies are required to detect other genes associated with resistance to amino-glycosides.

Antimicrobial Susceptibilities of Oral Isolates of Abiotrophia and Granulicatella According to the Consensus Guidelines for Fastidious Bacteria.

Background: The genera Abiotrophia and Granulicatella, previously known as nutritionally variant streptococci (NVS), are fastidious bacteria requiring vitamin B6 analogs for growth. They are members of human normal oral microbiota, and are supposed to be one of the important pathogens for so-called “culture-negative” endocarditis. Methods: The type strains and oral isolates identified, by using both phenotypic profiles and the DNA–DNA hybridization method, were examined for susceptibilities to 15 antimicrobial agents including penicillin (benzylpenicillin, ampicillin, amoxicillin, and piperacillin), cephem (cefazolin, ceftazidime, ceftriaxone, and cefaclor), carbapenem (imipenem), aminoglycoside (gentamicin), macrolide (erythromycin), quinolone (ciprofloxacin), tetracycline (minocycline), glycopeptide (vancomycin), and trimethoprim-sulfamethoxazole complex. The minimum inhibitory concentration and susceptibility criterion were determined, according to the consensus guideline from the Clinical and Laboratory Standards Institute. Results: Isolates of Abiotrophia defectiva were susceptible to ampicillin, amoxicillin ceftriaxone, cefaclor, imipenem, ciprofloxacin, and vancomycin. Isolates of Granulicatella adiacens were mostly susceptible to benzylpenicillin, ampicillin, amoxicillin, cefazolin, ceftriaxone, imipenem, minocycline, and vancomycin. The susceptibility profile of Granulicatella elegans was similar to that of G. adiacens, and the susceptibility rate was higher than that of G. adiacens. Conclusions: Although Abiotrophia and Granulicatella strains are hardly distinguishable by their phenotypic characteristics, their susceptibility profiles to the antimicrobial agents were different among the species. Species-related differences in susceptibility of antibiotics should be considered in the clinical treatment for NVS related infections.

Antibiotic Resistance Trends of Gram-negative Bacteria Most Frequently Isolated from Inpatients in a Tertiary Care Hospital in Sana'a, Yemen

Objective: To determine the trends of antibiotic resistance of Gram-negative bacteria, most frequently isolated from inpatients at the University of Science and Technology Hospital (USTH) in Sana'a, Yemen.
 Methods: A retrospective, cross-sectional study on the antibiotic resistance of Gram-negative bacteria most frequently isolated from respiratory tract, pus, urine, blood and other types of specimens from inpatients admitted to the USTH. Data were retrieved from the hospital records of culture-positive inpatients in the period from January 2006 to December 2013, and annual trends of resistance were compared using chi-square test for trends at P values < 0.05.
 Results: Of 2005 Gram-negative bacterial isolates in the period from 2006 to 2013, the most frequently isolated species were Escherichia coli (41.6%), Acinetobacter species (26.7%), Klebsiella species (21.0%) and Pseudomonas aeruginosa (10.6%). Amikacin and carbapenems were the most active drugs against E. coli, with a decrease in the susceptibility of this species to the third- and fourth-generation cephalosporins and a variable resistance rate to quinolones that significantly increased in 2013. Acinetobacter species susceptibility to most antibiotics decreased significantly over the years of the study, where polymyxin B was the only one found to be effective against this species. On the other hand, the trend of Klebsiella species resistance to imipenem, piperacillin-tazobactam, cefepime, ceftazidime increased over the years of the study. Susceptibility of Klebsiella species to ciprofloxacin, levofloxacin and moxifloxacin showed fluctuations, while the susceptibility of aminoglycosides (amikacin and gentamicin) and ampicillin-sulbactam showed no difference. The resistance of P. aeruginosa to the majority of antibiotics was not dramatically changed over the years of the study period, but gentamicin resistance rate was considerably dropped from 77.8% in 2008 to 25.9% in 2013.
 Conclusions: Of the most frequently isolated Gram-negative bacteria in Sana'a, Acinetobacter species has the highest resistance rate to the most commonly used antibiotics, where only polymyxin B is effective against this species. P. aeruginosa shows an unchanging rate of resistance to antibiotics in the USTH despite being quite resistant to antibiotics on a global scale, which could be attributed to the smaller number of P. aeruginosa isolates tested over the study period. Further large-scale studies on the trends of antibiotic resistance rates in hospital-based settings and the best ways to counteract such resistance in Yemen are recommended.

Antibiotic Resistance Trends of Gram-negative Bacteria Most Frequently Isolated from Inpatients in a Tertiary Care Hospital in Sana'a, Yemen

Objective: To determine the trends of antibiotic resistance of Gram-negative bacteria, most frequently isolated from inpatients at the University of Science and Technology Hospital (USTH) in Sana'a, Yemen.
 Methods: A retrospective, cross-sectional study on the antibiotic resistance of Gram-negative bacteria most frequently isolated from respiratory tract, pus, urine, blood and other types of specimens from inpatients admitted to the USTH. Data were retrieved from the hospital records of culture-positive inpatients in the period from January 2006 to December 2013, and annual trends of resistance were compared using chi-square test for trends at P values < 0.05.
 Results: Of 2005 Gram-negative bacterial isolates in the period from 2006 to 2013, the most frequently isolated species were Escherichia coli (41.6%), Acinetobacter species (26.7%), Klebsiella species (21.0%) and Pseudomonas aeruginosa (10.6%). Amikacin and carbapenems were the most active drugs against E. coli, with a decrease in the susceptibility of this species to the third- and fourth-generation cephalosporins and a variable resistance rate to quinolones that significantly increased in 2013. Acinetobacter species susceptibility to most antibiotics decreased significantly over the years of the study, where polymyxin B was the only one found to be effective against this species. On the other hand, the trend of Klebsiella species resistance to imipenem, piperacillin-tazobactam, cefepime, ceftazidime increased over the years of the study. Susceptibility of Klebsiella species to ciprofloxacin, levofloxacin and moxifloxacin showed fluctuations, while the susceptibility of aminoglycosides (amikacin and gentamicin) and ampicillin-sulbactam showed no difference. The resistance of P. aeruginosa to the majority of antibiotics was not dramatically changed over the years of the study period, but gentamicin resistance rate was considerably dropped from 77.8% in 2008 to 25.9% in 2013.
 Conclusions: Of the most frequently isolated Gram-negative bacteria in Sana'a, Acinetobacter species has the highest resistance rate to the most commonly used antibiotics, where only polymyxin B is effective against this species. P. aeruginosa shows an unchanging rate of resistance to antibiotics in the USTH despite being quite resistant to antibiotics on a global scale, which could be attributed to the smaller number of P. aeruginosa isolates tested over the study period. Further large-scale studies on the trends of antibiotic resistance rates in hospital-based settings and the best ways to counteract such resistance in Yemen are recommended.

Temporal trends and predictors of antimicrobial resistance among Staphylococcus spp. isolated from canine specimens submitted to a diagnostic laboratory.

BackgroundResistance to commonly used antimicrobials is a growing concern in both human and veterinary medicine. Understanding the temporal changes in the burden of the problem and identifying its determinants is important for guiding control efforts. Therefore, the objective of this study was to investigate temporal patterns and predictors of antimicrobial resistance among Staphylococcus spp. isolated from canine specimens submitted to the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL) between 1993 and 2009.MethodsRetrospective data of 4,972 Staphylococcus isolates assessed for antimicrobial susceptibility using the disk diffusion method at the UKVDL between 1993 and 2009 were included in the study. Temporal trends were assessed for each antimicrobial using the Cochran-Armitage trend test. Logistic regression models were used to investigate predictors of antimicrobial resistance (AMR) and multidrug resistance (MDR).ResultsA total of 68.2% (3,388/4,972) Staphylococcus isolates were S. intermedius group (SIG), 18.2% (907/4,972) were coagulase-negative staphylococci (CoNS), 7.6% (375/4,972) were S. aureus, 5.8% (290/4,972) were S. hyicus, and S. schleiferi subsp. coagulans comprised 0.2% (12/4,972) of the isolates. The overall percentage of AMR and MDR were 77.2% and 25.6%, respectively. The highest levels of AMR were seen in CoNS (81.3%; 737/907), S. aureus (80.5%; 302/375), and SIG (77.6%; 2,629/3388). The lowest levels of AMR were observed in S. hyicus (57.9%; 168/290) and S. schleiferi subsp. coagulans (33.3%; 4/12). Overall, AMR and MDR showed significant (p<0.001) decreasing temporal trends. Significant temporal trends (both increasing and decreasing) were observed among 12 of the 16 antimicrobials covering 6 of the 9 drug classes assessed. Thus, significant increasing temporal trends in resistance were observed to β-lactams (p<0.001) (oxacillin, amoxicillin-clavulanate, cephalothin, and penicillin (p = 0.024)), aminoglycosides (p<0.001) (gentamicin, and neomycin), bacitracin (p<0.001), and enrofloxacin (p<0.001). In contrast, sulfonamide (p<0.001) (sulfadiazin) and tetracycline (p = 0.010) resistant isolates showed significant decreasing temporal trends in AMR. Staphylococcus spp., geographic region, and specimen source were significant predictors of both AMR and MDR.ConclusionsAlthough not unexpected nor alarming, the high levels of AMR to a number of antimicrobial agents and the increasing temporal trends are concerning. Therefore, continued monitoring of AMR among Staphylococcus spp. is warranted. Future studies will need to identify local factors responsible for the observed geographic differences in risk of both AMR and MDR.

Molecular characterization of extended spectrum β -lactamases enterobacteriaceae causing lower urinary tract infection among pediatric population

BackgroundThe β-lactam antibiotics have traditionally been the main treatment of Enterobacteriaceae infections, nonetheless, the emergence of species producing β- Lactamases has rendered this class of antibiotics largely ineffective. There are no published data on etiology of urinary tract infections (UTI) and antimicrobial resistance profile of uropathogens among children in Qatar. The aim of this study is to determine the phenotypic and genotypic profiles of antimicrobial resistant Enterobacteriaceae among children with UTI in Qatar.MethodsBacteria were isolated from 727 urine positive cultures, collected from children with UTI between February and June 2017 at the Pediatric Emergency Center, Doha, Qatar. Isolated bacteria were tested for antibiotic susceptibility against sixteen clinically relevant antibiotics using phoenix and Double Disc Synergy Test (DDST) for confirmation of extended-spectrum beta-lactamase (ESBL) production. Existence of genes encoding ESBL production were identified using polymerase chain reaction (PCR). Statistical analysis was done using non-parametric Kappa statistics, Pearson chi-square test and Jacquard’s coefficient.Results201 (31.7%) of samples were confirmed as Extended Spectrum β -Lactamases (ESBL) Producing Enterobacteriaceae. The most dominant pathogen was E. coli 166 (83%) followed by K. pneumoniae 22 (11%). Resistance was mostly encoded by bla CTX-M (59%) genes, primarily bla CTX-MG1 (89.2%) followed by bla CTX-MG9 (7.7%). 37% of isolated bacteria were harboring multiple bla genes (2 genes or more). E. coli isolates were categorized into 11 clusters, while K. pneoumoniae were grouped into five clonal clusters according to the presence and absence of seven genes namely bla TEM, bla SHV, bla CTX-MG1, bla CTX-MG2, bla CTX-MG8 bla CTX-MG9,bla CTX-MG25.ConclusionsOur data indicates an escalated problem of ESBL in pediatrics with UTI, which mandates implementation of regulatory programs to reduce the spread of ESBL producing Enterobacteriaceae in the community. The use of cephalosporins, aminoglycosides (gentamicin) and trimethoprim/sulfamethoxazole is compromised in Qatar among pediatric population with UTI, leaving carbapenems and amikacin as the therapeutic option for severe infections caused by ESBL producers.

Association between gentamicin resistance and stress tolerance in water isolates of Ralstonia pickettii and R. mannitolilytica.

Members of the species Ralstonia pickettii and R. mannitolilytica, although ubiquitous and lacking major virulence factors, have been associated with nosocomial outbreaks. Tolerance to metals, antibiotics, and disinfectants may represent an advantage for their ubiquity and opportunistic pathogenic potential. In this study, we compared five strains that differed on the origin (hospital effluent, tap water, mineral water) and in the susceptibility to aminoglycosides, regarding their tolerance to metals and disinfection. The growth kinetics and biofilm formation capacity were tested in four R. pickettii strains and one R. mannitolilytica at sub-inhibitory concentrations of aminoglycosides or arsenite. The survival to UV radiation, chlorine, or hydrogen peroxide was also compared in aminoglycoside resistant and susceptible strains. Aminoglycoside-resistant strains presented a higher tolerance to arsenite than the susceptible ones and either aminoglycosides or arsenite was observed to stimulate the biofilm formation. Sub-inhibitory concentrations of the aminoglycoside gentamicin or arsenite significantly decreased the growth rate and yield, but only arsenite caused a significant increase of the lag phase. Hydrogen peroxide presented higher disinfection effectiveness against aminoglycoside susceptible than against resistant strains, an effect that was not observed for UV or chlorine. Although this conclusion needs validation based on a larger number of isolates, including clinical, the results suggest that aminoglycoside resistance may be associated with traits that influence Ralstonia spp. fitness in the environment.

Does ivacaftor interfere with the antimicrobial activity of commonly used antibiotics against Pseudomonas aeruginosa?-Results of an in vitro study.

Ivacaftor is a novel potentiator of defective cystic fibrosis transmembrane conductance regulator (CFTR) protein, which corrects the gating defect and increases ion-function of activated cell-surface CFTR. Bacteria also regulate their physiology through ion channels. However, little is known about the potential effects of ivacaftor on bacterial ion channels, which, in turn, may have a potential effect on transport across the bacterial cell membrane. Therefore, any change in the ability to transport molecules across cell membranes in bacteria could have an important impact on bacterial transport physiology. One area where this could be particularly important is in the movement of antibiotics, both into and out of the bacterial cell. An in vitro study was therefore performed to examine the influence of ivacaftor at therapeutic concentration on antibiotic susceptibility of 11 commonly used anti-pseudomonal antibiotics against a population of clinical Pseudomonas aeruginosa [PA], from CF and non-CF sources. Pseudomonas aeruginosa (n=80; including 70 ivacaftor-naïve clinical PA from sputa from adult CF patients and 10 control PA from non-CF clinical blood culture sources) were examined. Antibiotic susceptibility was determined by standard disc diffusion assay using CLSI criteria and measuring zone size (mm), against four classes of anti-pseudomonal antibiotics, including beta-lactams (temocillin, ceftazidime, piperacillin/tazobactam, imipenem, meropenem and aztreonam), aminoglycosides (gentamicin, tobramycin, amikacin), fluoroquinolone (ciprofloxacin) and polymyxin (colistin), in the absence and presence of ivacaftor (5μmol/L), as previously determined. In addition, all CF and non-CF PA were examined phenotypically in vitro, as previously described, for changes linked to bacterial virulence, including (i) growth density (ii) pigmentation, (iii) presence of adhesins and (iv) change to mucoidy, in the presence/absence of ivacaftor at therapeutic concentration. Antibiotic susceptibility did not decrease significantly with any of the antibiotics examined with CF PA isolates or with non-CF PA control organisms. There was a statistically significant increase in zone size (CF PA and amikacin, gentamicin, temocillin and ciprofloxacin; Non-CF PA and amikacin, gentamicin and aztreonam). However, at a population level, this did not translate into a shift in CLSI category to a more susceptible phenotype. None of the PA isolates examined were susceptible to ivacaftor alone, and additionally, no changes were noted with the four phenotypic parameters examined in the presence of ivacaftor. This study showed that antibiotic susceptibility of commonly used anti-pseudomonal antibiotics was not negatively affected by ivacaftor, in a population of ivacaftor-naive P.aeruginosa.

Differentiation and Induced Sensorial Alteration of the Coronal Organ in the Asexual Life of a Tunicate.

Tunicates, the sister group of vertebrates, possess a mechanoreceptor organ, the coronal organ, which is considered the best candidate to address the controversial issue of vertebrate hair cell evolution. The organ, located at the base of the oral siphon, controls the flow of seawater into the organism and can drive the "squirting" reaction, i.e., the rapid body muscle contraction used to eject dangerous particles during filtration. Coronal sensory cells are secondary mechanoreceptors and share morphological, developmental, and molecular traits with vertebrate hair cells. In the colonial tunicate Botryllus schlosseri, we described coronal organ differentiation during asexual development. Moreover, we showed that the ototoxic aminoglycoside gentamicin caused morphological and mechanosensorial impairment in coronal cells. Finally, fenofibrate had a strong protective effect on coronal sensory cells due to gentamicin-induced toxicity, as occurs in vertebrate hair cells. Our results reinforce the hypothesis of homology between vertebrate hair cells and tunicate coronal sensory cells.

Occurrence and molecular composition of methicillin-resistant Staphylococcus aureus isolated from ocular surfaces of horses presented with ophthalmologic disease

Severe infections due to methicillin-resistant Staphylococcus aureus (MRSA) have been increasingly recognized in virtually all fields of veterinary medicine. Our objective was to study the occurrence, phylogenetic relationships and antimicrobial resistance properties of MRSA isolated from ocular surfaces of horses prior to invasive procedures. Within a 49-week sampling period, ocular swabs obtained from 46 eyes of 44 horses, including eyes with clinical signs of conjunctivitis/blepharitis, keratitis or uveitis were screened for the presence of S. aureus. As a result, seven samples were positive for S. aureus (15.2%), with six of them being classified as MRSA (13%). In addition, all isolates were resistant or showed reduced susceptibility to tetracyclines, the aminoglycosides gentamicin and kanamycin, fluoroquinolones, and the combination sulfamethoxazole/trimethoprim. Since a very close relationship between the MRSA isolates was assumed after pulsed-field gel electrophoresis employing the restriction endonuclease ApaI, whole genome sequencing (WGS) was used to shed more light on the phylogenetic relationships and the molecular composition of all MRSA isolates. Analysis of WGS data revealed closely related MRSA belonging to sequence type 398, spa type t011 and dru type dt10q, harboring an SCCmec IV element and the Staphylococcus aureus pathogenicity island SaPIbov5. Moreover, all MRSA were positive for a beta-hemolysin converting phage carrying genes of the immune evasion cluster (IEC). Since cases of eye infections due to MRSA were often associated with fatal outcomes, more research is needed with respect to the origin of MRSA isolated from ocular surfaces to implement sufficient barrier and infection control measures.

Readthrough of ACTN3 577X nonsense mutation produces full-length α-actinin-3 protein

The ACTN3 gene encodes α-actinin-3 protein, which stabilizes the contractile apparatus at the Z-line in skeletal muscle cell fast fibers. A nonsense mutation of the arginine (R) at the codon for amino acid 577 of the ACTN3 gene generates a premature termination codon (PTC) and produces the R577X polymorphism in humans (X specifies translational termination). The ACTN3 577X genotype abolishes α-actinin-3 protein production due to targeted degradation of the mutant transcript by the cellular nonsense-mediated mRNA decay (NMD) system, which requires mRNA splicing. In humans, α-actinin-3 deficiency can decrease sprinting and power performance as well as skeletal muscle mass and strength. Here we investigated whether suppression of the in-frame PTC induced by treatment with the aminoglycosides gentamicin and G418 that promote termination codon readthrough could allow production of full-length α-actinin-3 protein from ACTN3 577X. We constructed expression plasmids encoding mature mRNA that lacks introns or pre-mRNA, which carries introns for the ACTN3 577X gene (X and Xpre, respectively) and transfected the constructs into HEK293 cells. Similar constructs for the ACTN3 577R gene were used as controls. HEK293 cells carrying the X gene, but not the Xpre gene, expressed exogenous truncated α-actinin-3 protein, indicating NMD-mediated suppression of exogenous Xpre expression. Cells treated with aminoglycosides produced exogenous full-length α-actinin-3 protein in X-transfected cells, but not in Xpre-transfected cells. The NMD inhibitor caffeine prevented suppression of Xpre expression and thereby induced production of full-length α-actinin-3 protein in the presence of aminoglycoside. Together these results indicate that the ACTN3 R577X polymorphism could be a novel target for readthrough therapy, which may affect athletic and muscle performance in humans.

Pseudomonas aeruginosa: Evaluation of Pathogen Burden and Drug-Resistance Trends in a Tertiary Care Hospital.

To evaluate the pathogen burden and antibiotic-resistance trends of Pseudomonas aeruginosa among hospitalised patients at a tertiary care hospital. Retrospective, hospital record-based, cross-sectional study. Microbiology Laboratory, Allama Iqbal Medical College/Jinnah Hospital, Lahore, from January 2014 to December 2016. A total of 5,960 samples were collected from clinically suspected cases of bacterial infections, admitted to the hospital. Microbial identification and antibiotic susceptibility pattern were carried out and analysed. Out of a total of 5,960 samples, Pseudomonas aeruginosa was isolated from 1,268 (21.2%) specimens. Department-wise isolation rate was n=600 (42.9%), n=268 (15.4%), n=201 (12.6%), and n=199 (16.0%) from intensive care unit (ICU), surgical units, medical units, and Gynae wards, respectively (p<0.0001). Sample-wise isolation rate was, wound swabs n=448 (35%), urine n=356 (28%), sputum n=187 (14 %), tracheal aspirate n=127 (10%), blood n=99 (7%), and broncho-alveolar lavage n=51 (4%) (p<0.0001). Drug-resistance pattern showed low rates for carbapenems(meropenem n=440 (35%), Imipenem n=436 (34%) and beta-lactam + beta-lactamase inhibitor combination (piperacillin+tazobactam n=437 (34%) while alarming rates were observed for cephalosporins (ceftazidime n=716 (56%), fluoroquinolones (ciprofloxacin n=690 (54%), cefoperazone+sulbactam n=685 (54%), aminoglycosides (gentamicin, n=669 (53%), amikacin n=608 (48%), and monobactams (aztreonam n=666 (52%). Decreasing trend was observed only for amikacin 63% to 37%, aztreonam showed similar pattern throughout, while there was an increasing trend of drug resistance in all groups of antibiotics. Emerging drug-resistant strains of Pseudomonas aeruginosa are probably linked to the injudicious use of antibiotics, leading to ineffective empirical therapy. Therefore, we suggest that culture and antimicrobial susceptibility testing should be done for targeted antimicrobial therapy against Pseudomonas aeruginosa.

From CLSI to EUCAST guidelines in the interpretation of antimicrobial susceptibility: What is the effect in our setting?

IntroductionImplementation of the breakpoints established in the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines in comparison with those of the Clinical and Laboratory Standards Institute (CLSI) means that the criteria for interpreting the susceptibility of some antimicrobials have been modified, resulting in changes in the reports of accumulated antibiotic susceptibility. MethodsThe effect of applying EUCAST breakpoints in 10,359 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus spp. was analysed. ResultsBy applying EUCAST breakpoints, most antimicrobial susceptibility percentages did not change or changed very slightly. However, a decrease in aminoglycoside susceptibility was observed in Gram-negative bacilli, mainly for amikacin and Pseudomonas aeruginosa (23.2%), although only 5.7% were completely resistant; a notably decrease in the percentage of isolates susceptible to aztreonam was also observed. There was also a marked increase in the number of Staphylococcus aureus strains resistant to clindamycin (51.5%) and aminoglycosides (gentamicin 43.1%). ConclusionsSwitching from CLSI to EUCAST criteria in some pathogens alters the percentages of resistance to several antimicrobials, and therefore the local epidemiology of the resistance. These changes should be implemented by a multidisciplinary group in order to analyse the influence of the new data on the empirical treatment protocols of each centre.

Induction of mitophagy in the HEI-OC1 auditory cell line and activation of the Atg12/LC3 pathway in the organ of Corti

Autophagy is a highly evolutionary conserved quality control defense mechanism within cells, which has also been implicated in cell death processes. In the mammalian inner ear, autophagy has been shown to play a role during early morphogenesis as well as in adult cochlear hair cells exposed to ototoxic insults. Mitophagy, a selective autophagic cell process targeting mitochondria, hasn't been studied in the inner ear so far. On this work, we searched for molecular indicators of mitophagy within House Ear Institute-Organ of Corti-1 (HEI-OC1) cells as well as in the organ of Corti (OC). We first tested for the expression of Pink1/Park2 mRNA in 5-day-old C57BL/6 mice's cochleae using RT-PCR. We focused on the induction of mitophagy in HEI-OC1 cells as well as in the OC and investigated a possible mitophagic potential of the aminoglycoside agent gentamicin. The induction of mitophagy in HEI-OC1 cells was detected by objectivizing the translocation of fluorescence-tagged LC3 to mitochondria using confocal microscopy after a 6-h incubation with a well-described mitochondrial uncoupler and mitophagy-inducing agent: carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Incubation with gentamicin generated no mitochondrial translocation of LC3. Protein levels of COXIV, Atg5/12 and LC3 were evaluated by an immunoblot analysis after a 24-h CCCP treatment as well as gentamicin. We demonstrated mitophagy after CCCP exposure in HEI-OC1 cells by showing a downregulation of COXIV. A downregulation of COXIV could also be visualized in the OC after CCCP. A significant oxygen consumption rate (OCR) changed in cells treated with CCCP as well as significant morphological changes of mitochondria by electron microscopy (EM) strengthen this assumption. Gentamicin exposure generated no impact on OCR or mitochondrial morphological changes by EM. Finally, we demonstrated changes in the expression of Atg12 and LC3 proteins in both the OC and HEI-OC1 cells after CCCP exposure but not after gentamicin. Our data indicate that gentamicin had no impact in the activation of mitophagy—neither in the HEI-OC1 cell line nor in the OC. Therefore, we speculate that mitophagic-independent mechanisms may underly aminoglycoside ototoxicity.

Synergistic Rescue of Nonsense Mutant Tumor Suppressor p53 by Combination Treatment with Aminoglycosides and Mdm2 Inhibitors.

The tumor suppressor gene TP53 is inactivated by mutation in a large fraction of human tumors. Around 10% of TP53 mutations are nonsense mutations that lead to premature termination of translation and expression of truncated unstable and non-functional p53 protein. Aminoglycosides G418 (geneticin) and gentamicin have been shown to induce translational readthrough and expression of full-length p53. However, aminoglycosides have severe side effects that limit their clinical use. Here, we show that combination treatment with a proteasome inhibitor or compounds that disrupt p53-Mdm2 binding can synergistically enhance levels of full-length p53 upon aminoglycoside-induced readthrough of R213X nonsense mutant p53. Full-length p53 expressed upon combination treatment is functionally active as assessed by upregulation of p53 target genes, suppression of cell growth, and induction of cell death. Thus, our results demonstrate that combination treatment with aminoglycosides and compounds that inhibit p53 degradation is synergistic and can provide significantly improved efficacy of readthrough when compared with aminoglycosides alone. This may have implications for future cancer therapy based on reactivation of nonsense mutant TP53.

ANTIMICROBIAL RESISTANCE OF Klebsiella pneumonia ISOLATES IN REPUBLIC OF SAKHA (YAKUTIA)

The aim of the study was to evaluate the susceptibility to antibiotics of Klebsiella pneumoniae isolates in the Republic of Sakha (Yakutia), according to the AMRmap database. Material and methods. Data of online platform AMRmap in 2010-2016 have been used for evaluation of the sensitivity of K. pneumoniae isolates to antibiotics. Results and discussion. K. pneumoniae isolates comprised 35,7 % of all bacterial nosocomial isolates ( n = 195) and 30,1 % of community-acquired isolates ( n = 382) respectively. Most of the isolates were insusceptible to common antibiotics: aminoglycosides (gentamicin, tobramycin, netilmicin) - 50-78 %; chloramphenicol - 36-43 %, cephalosporins III-V generation (ceftazidime, cefotaxime, zepefin, ceftarolin) - 83-100 %, penicillin drugs with inhibitor of beta-lactamases (ticarcillin-clavulanate, amoxicillin-clavulanate) - 83-98 % monobactams (aztreonam) - 87-95 %, semi-synthetic penicillins of broad spectrum of action (ampicillin) - 100 %, sulfonamides (trimethoprim-sulfamethoxazole) - 53-76 %, fluoroquinolones (ciprofloxacin) - 77 %. At present the next preparations can be effective against K. pneumonia: amikacin (sensitivity 96-100 %), ceftazidime-avibactam (95-97 %), tigecycline (97-100 %), carbapenems (87-97 %), in some cases, phosphomycin (77 %) and piperacillin-tazobactam (64-71 %). Conclusion. The spectrum of drugs for the treatment of infections caused by K. pneumoniae is extremely limited. The further spread of resistance to carbapenems poses a threat of ineffectiveness in the treatment of severe infections. In these conditions, measures are needed to limit the unjustified use of carbapenems. The indicators of resistance of K. pneumoniae to antibiotics should be taken into account not only for the effective treatment of patients, but also for optimizing the costs of regional health care, for example when planning procurement of medicines for hospitals

Physical Compatibility of Meropenem and Vaborbactam With Select Intravenous Drugs During Simulated Y-site Administration

PurposeMeropenem/vaborbactam is a novel intravenous antibiotic combining the carbapenem, meropenem, with a novel β-lactamase inhibitor, vaborbactam. Meropenem/vaborbactam is administered as a 3-hour infusion given every 8 hours, thereby potentially restricting an intravenous line for 9 h/d. Intravenous medications may be given concurrently via Y-site when compatibility data are available. Herein, physical compatibility was determined for the identification which medications can be coadministered with meropenem/vaborbactam via Y-site. MethodsY-site administration was simulated in vitro by admixing 5 mL of meropenem 8 mg/mL and vaborbactam 8 mg/mL with an equal volume of 88 other diluted intravenous medications, including 34 antimicrobials. All other medications were diluted with 0.9% sodium chloride to the upper range of concentrations considered standard for intravenous infusion. Visual inspection, turbidity measurement, and pH measurement were performed prior to admixture, directly after admixture, and at time points up to 3 hours after admixture. FindingsOf the 88 medications tested, meropenem/vaborbactam was compatible with 73 (83%), including many antibiotics such as aminoglycosides (amikacin, gentamicin, and tobramycin), colistin, fosfomycin, linezolid, tedizolid, tigecycline, and vancomycin. Physical incompatibility was observed with albumin, amiodarone, anidulafungin, calcium chloride, caspofungin, ceftaroline, ciprofloxacin, daptomycin, diphenhydramine, dobutamine, isavuconazole, midazolam, nicardipine, ondansetron, and phenytoin. ImplicationsThe majority of intravenous medications tested were found to be physically compatible with meropenem/vaborbactam. These data will help pharmacists and nurses to improve line access in patients receiving meropenem/vaborbactam.

Proline-rich antimicrobial peptides show a long-lasting post-antibiotic effect on Enterobacteriaceae and Pseudomonas aeruginosa

Proline-rich antimicrobial peptides (PrAMPs) represent a promising class of potential therapeutics to treat multiresistant infections. They inhibit bacterial protein translation at the 70S ribosome by either blocking the peptide-exit tunnel (oncocin type) or trapping release factors (apidaecin type). Besides direct concentration-dependent antibacterial effects, the post-antibiotic effect (PAE) is the second most important criterion of antimicrobial pharmacodynamics to be determined in vitro. Here, PAEs of 10 PrAMPs and three antibiotics against three Escherichia coli strains, Klebsiella pneumoniae ATCC 10031 and Pseudomonas aeruginosa ATCC 27853 were studied after 1 h of exposure. A robust high-throughput screening to determine PAEs was established, i.e. liquid handling by a 96-channel pipetting system and continuous incubation and absorbance measurement in a microplate reader. Prolonged PAEs (≥4 h) were detected for all peptides at their MIC values against all strains; PAEs were even >10 h for Api88, Api137, Bac7(1-60) and A3-APO. The PAEs increased further at 4 × MIC. Aminoglycosides gentamicin and kanamycin usually showed lower PAEs (≤4 h) at MIC, but PAEs increased to > 10 h at 4 × MIC. Bacteriostatic chloramphenicol exhibited the shortest PAEs (<4 h). The PAEs of PrAMPs studied against Enterobacteriaceae and P. aeruginosa for the first time were typically 4-fold stronger than for conventional antibiotics. Together with their fast and irreversible uptake by bacteria, the observed prolonged PAE of PrAMPs helps to explain their high in vivo efficacy despite unfavourable pharmacokinetics.

Genotyping and drug susceptibility testing of mycobacterial isolates from population-based tuberculosis prevalence survey in Ghana

BackgroundMycobacterium tuberculosis complex (MTBC) and Non-tuberculosis Mycobacterium (NTM) infections differ clinically, making rapid identification and drug susceptibility testing (DST) very critical for infection control and drug therapy. This study aims to use World Health Organization (WHO) approved line probe assay (LPA) to differentiate mycobacterial isolates obtained from tuberculosis (TB) prevalence survey in Ghana and to determine their drug resistance patterns.MethodsA retrospective study was conducted whereby a total of 361 mycobacterial isolates were differentiated and their drug resistance patterns determined using GenoType Mycobacterium Assays: MTBC and CM/AS for differentiating MTBC and NTM as well MTBDRplus and NTM-DR for DST of MTBC and NTM respectively.ResultsOut of 361 isolates, 165 (45.7%) MTBC and 120 (33.2%) NTM (made up of 14 different species) were identified to the species levels whiles 76 (21.1%) could not be completely identified. The MTBC comprised 161 (97.6%) Mycobacterium tuberculosis and 4 (2.4%) Mycobacterium africanum. Isoniazid and rifampicin monoresistant MTBC isolates were 18/165 (10.9%) and 2/165(1.2%) respectively whiles 11/165 (6.7%) were resistant to both drugs. Majority 42/120 (35%) of NTM were M. fortuitum. DST of 28 M. avium complex and 8 M. abscessus complex species revealed that all were susceptible to macrolides (clarithromycin, azithromycin) and aminoglycosides (kanamycin, amikacin, and gentamicin).ConclusionOur research signifies an important contribution to TB control in terms of knowledge of the types of mycobacterium species circulating and their drug resistance patterns in Ghana.