Clinical Isolates Of Gram-negative Bacteria Research Articles

Characterization of aminoglycoside resistance genes in multidrug-resistant Klebsiella pneumoniae collected from tertiary hospitals during the COVID-19 pandemic.

Since the peak of the coronavirus disease 2019 (COVID-19) pandemic, concerns around multidrug-resistant (MDR) bacterial pathogens have increased. This study aimed to characterize aminoglycoside resistance genes in MDR Klebsiella pneumoniae (K. pneumoniae) collected during the COVID-19 pandemic. A total of 220 clinical isolates of gram-negative bacteria were collected from tertiary hospitals in Makkah, Saudi Arabia, between April 2020 and January 2021. The prevalence of K. pneumoniae was 40.5%; of the 89 K. pneumoniae isolates, MDR patterns were found among 51 (57.3%) strains. The MDR isolates showed elevated resistance rates to aminoglycoside agents, including amikacin (100%), gentamicin (98%), and tobramycin (98%). PCR assays detected one or more aminoglycoside genes in 42 (82.3%) MDR K. pneumoniae strains. The rmtD gene was the most predominant gene (66.7%; 34/51), followed by aac(6')-Ib and aph(3')-Ia (45.1%; 23/51). The aac(3)-II gene was the least frequent gene (7.8%; 4/51) produced by our isolates. The rmtC gene was not detected in the studied isolates. Our findings indicated a high risk of MDR bacterial infections through the COVID-19 outbreak. Therefore, there is a need for continuous implementation of effective infection prevention control (IPC) measures to monitor the occurrence of MDR pathogens and the emergence of MDR bacterial infections through the COVID-19 outbreak.

In silico and In vitro Assessment of Antibacterial Activity, LPS Binding Affinity, and Toxicity of the GKY25 Peptide.

Extensively and multi-drug resistant isolates of bacteria (MDR, XDR) have caused significant health problems and are responsible for high morbidity and mortality as well. In this critical condition, the discovery, design, or development of new antibiotics is of great concern. According to this necessity, antimicrobial peptides (AMPs) suggested as promising agents. Accordingly, this study aims to evaluate the GKY25 peptide to develop its future antibacterial applications as well as confirmation of LPS neutralization. Predictions of 3D structure and helical wheel projection analysis of the peptide were performed by ITASSER and Heliquest servers. Binding affinity and antibacterial activity were performed using molecular docking and CAMPR4, respectively, followed by experimental binding assay as well as in vitro antibacterial assay. GKY25 was predicted as an alpha-helical peptide, and its helicity showed probable projection of hydrophobic and positively-charged amino acid residues. Docking studies showed binding affinity of GKY25 peptide to gram-positive and outer and inner gram-negative bacterial membranes as -5.7, -6.8, and -4 kcal/mole, respectively. CAMPR4 analysis predicted the peptide as an AMP. Experimental binding assay showed that the peptide binds LPS immediately and their interaction was observed at 274 nm. Gathering all in silico and in vitro data together, GKY25 is a good drug lead that could be examined further using clinical isolates of gram-negative bacteria in vitro.

The association between biofilm formation ability and antibiotic resistance phenotype in clinical isolates of gram-negative bacteria: a cross-sectional study

Backgrounds: Antimicrobial-resistant pathogens that form biofilms cause treatment failure, increased mortality, and morbidity. Gram-negative bacteria (GNB) infections have become an emerging issue in antimicrobial resistance. This study analyzes the association between GNB biofilm formation ability and the antibiotic resistance phenotype in GNB clinical isolates. Methods: From June to August 2022, this cross-sectional study was conducted at Dr. Soetomo General Academic Hospital. Isolates derived from clinical specimens, including blood, urine, and respiratory tract specimens, were identified for antibiotic resistance phenotypes along with a quantitative biofilm formation assay. The antibiotic resistance phenotype was studied in relation to the GNB isolates' ability for biofilm formation. Results: Of the 271 isolates studied, 95 (35.1%) were non-MDR, 143 (52.8%) were MDR, and 33 (12.2%) were XDR. On the biofilm formation assay, 34 (12.5%) were non-biofilm producers, 237 (87.5%) were biofilm producers, consisting of 101 (37.3%) weak, 131 (48.3%) moderate, and 5 (1.8%) strong biofilm producers. Biofilm formation and antibiotic resistance in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii were not significantly associated (p > 0.05). However, Escherichia coli isolates resistant to the antibiotics amikacin, imipenem, meropenem, and amoxicillin-clavulanate were found to have a higher predisposition to form biofilms. Klebsiella pneumoniae isolates resistant to gentamycin were also positively associated with biofilm formation. Conclusions: Gram-negative bacteria's ability to form biofilms is not associated with their antibiotic resistance phenotype; therefore, routine biofilm monitoring is critical for enhancing the quality of treatment strategy for biofilm-associated infection.

Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria.

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-β-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four β-lactamase genes (bla KPC, bla NDM-1, bla IMP-1 group, and bla VIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six β-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four β-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four β-lactamases.

The Antibacterial Activity of Kaempferol Combined with Colistin against Colistin-Resistant Gram-Negative Bacteria.

Social hygiene is seriously threatened by the rise in colistin (COL) resistance against Gram-negative bacteria (GNB). With resistance to last-line antibiotics such as COL becoming more common, it is imperative to identify alternative treatment options. In our work, we sought to determine if COL plus kaempferol (KP) present synergistic effects on the antibacterial and antibiofilm activities against colistin-resistant (Col-R) GNB in vivo and in vitro. Twenty-four Col-R GNB were collected as the experimental strains. The synergistic activity of COL and KP was evaluated by checkerboard method, time-killing assays, and the Galleria mellonella experiment. The antibiofilm effectiveness of the COL/KP combination against Col-R GNB was assessed using biofilm inhibition and eradication assays and scanning electron microscopy (SEM). Cytotoxicity tests were performed to detect the toxicity of KP monotherapy or combination therapy. There is synergistic antibacterial activity of COL and KP combination in vitro. KP combined with COL could inhibit the formation of bacterial biofilms. The amalgamation of COL and KP considerably reduced the amount of bacteria in the biofilm, according to the SEM findings. The COL/KP combination improved the survivorship of infected larvae in the G. mellonella in vivo infection model. In addition, the combination of KP and COL showed no cytotoxicity at synergistic combined concentrations according to cytotoxicity assays. This represents the first account of the antibacterial and antibiofilm activities of KP in combination with COL against Col-R GNB. Therefore, our results may provide an effective alternative route to combat Col-R GNB infections. IMPORTANCE COL is one of the few antibiotics effective against clinical isolates of GNB. However, in recent years, GNB resistance to colistin has been increasing. As a result, the combined application of colistin in conjunction with nonantibacterial medications has garnered considerable interest. In this work, the KP/COL combination showed effective antibacterial and antibiofilm activities in vitro and in vivo. The synergistic effect of combined application may be attributed to membrane permeability. Due to the low cytotoxicity of the combined concentration, the combination exhibits a promising future for use in clinical anti-infection treatments. This finding might broaden the potential applications for COL.

Antimicrobial activity of cefepime/zidebactam (WCK 5222), a β-lactam/β-lactam enhancer combination, against clinical isolates of Gram-negative bacteria collected worldwide (2018-19).

Zidebactam, a bicyclo-acyl hydrazide β-lactam 'enhancer' antibiotic, in combination with cefepime (WCK 5222) is under clinical development for the treatment of resistant Gram-negative infections. To evaluate the in vitro activity of cefepime/zidebactam and comparators against 24 220 Gram-negative bacteria. Organisms were consecutively collected in 2018-19 from 137 medical centres located in the USA (n = 9140), Western Europe (W-EU; n = 5929), Eastern Europe (E-EU; n = 3036), the Asia-Pacific region (APAC; n = 3791) and Latin America (LATAM; n = 2324). The isolates were susceptibility tested using the broth microdilution method as part of the SENTRY Program. Cefepime/zidebactam was tested at a 1:1 ratio. Cefepime/zidebactam was highly active against Enterobacterales (MIC50/90 0.03/0.25 mg/L; 99.9% inhibited at ≤8 mg/L) and retained potent activity against carbapenem-resistant Enterobacterales (CRE) isolates (97.8% inhibited at ≤8 mg/L). CRE rates varied widely from 1.1% in the USA to 1.9% in W-EU, 3.6% in APAC and 14.6% in E-EU (3.9% overall). The most common carbapenemase genes observed overall were blaKPC (37.6% of CRE), blaOXA-48-like (30.0%) and blaNDM (23.8%). Resistance to ceftazidime/avibactam among CRE was elevated in APAC (64.8%), E-EU (25.5%) and LATAM (20.7%). Against Pseudomonas aeruginosa, cefepime/zidebactam inhibited 99.2% of isolates at ≤8 mg/L and susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam was lowest in E-EU (83.9% and 82.0%, respectively). Cefepime/zidebactam exhibited good activity against Stenotrophomonas maltophilia (80.0% inhibited at ≤8 mg/L) and Burkholderia cepacia (89.4% inhibited at ≤8 mg/L). Cefepime/zidebactam demonstrated potent in vitro activity against a large worldwide collection of contemporary clinical isolates of Gram-negative bacteria.

Resveratrol Reverts Tolerance and Restores Susceptibility to Chlorhexidine and Benzalkonium in Gram-Negative Bacteria, Gram-Positive Bacteria and Yeasts.

The spread of microorganisms causing health-care associated infection (HAI) is contributed to by their intrinsic tolerance to a variety of biocides, used as antiseptics or disinfectants. The natural monomeric stilbenoid resveratrol (RV) is able to modulate the susceptibility to the chlorhexidine digluconate (CHX) biocide in Acinetobacter baumannii. In this study, a panel of reference strains and clinical isolates of Gram-negative bacteria, Gram-positive bacteria and yeasts were analyzed for susceptibility to CHX and benzalkonium chloride (BZK) and found to be tolerant to one or both biocides. The carbonyl cyanide m-chlorophenylhydrazine protonophore (CCCP) efflux pump inhibitor reduced the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of CHX and BZK in the majority of strains. RV reduced dose-dependently MIC and MBC of CHX and BZK biocides when used as single agents or in combination in all analyzed strains, but not CHX MIC and MBC in Pseudomonas aeruginosa, Candida albicans, Klebsiella pneumoniae, Stenotrophomonas maltophilia and Burkholderia spp. strains. In conclusion, RV reverts tolerance and restores susceptibility to CHX and BZK in the majority of microorganisms responsible for HAI. These results indicates that the combination of RV, CHX and BZK may represent a useful strategy to maintain susceptibility to biocides in several nosocomial pathogens.

W090 Effect of carbapenemase, qace, qace⊗1, and cepa genes on chlorhexidine sensitivity in clinical isolates of gram-negative bacteria with multiple resistance

W090 Effect of carbapenemase, qace, qace⊗1, and cepa genes on chlorhexidine sensitivity in clinical isolates of gram-negative bacteria with multiple resistance

In Vitro Potency and Spectrum of the Novel Polymyxin MRX-8 Tested against Clinical Isolates of Gram-Negative Bacteria.

The polymyxins display excellent in vitro antimicrobial activity against most Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii isolates, but their clinical utility has been limited because of class-specific toxicity problems. Therefore, new polymyxin analogs with improved safety properties are needed to combat serious infections caused by resistant Gram-negative pathogens. MRX-8 is a novel polymyxin B analog that displays reduced toxicity in in vitro and animal assays and is currently being evaluated in a phase 1 clinical trial. In this nonclinical study, the in vitro potency and spectrum of MRX-8 and comparators were evaluated against a large set of Gram-negative clinical isolates collected in the United States in 2017 to 2020. MRX-8, colistin, and polymyxin B exhibited nearly identical antimicrobial activities against the Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii isolate sets. MRX-8 MIC50 and MIC90 values were 0.12 and 0.25 mg/L, respectively, for the set of Enterobacterales isolates not intrinsically resistant to colistin and 0.5 and 1 mg/L, respectively, against both the A. baumannii and P. aeruginosa isolate sets. All three polymyxin-class compounds retained activity against meropenem-resistant and multidrug-resistant isolate subsets but were inactive against isolates displaying acquired or intrinsic resistance to polymyxins. These results support the continued development of MRX-8 to treat serious Gram-negative infections.

Evaluation of phenotypic and genotypic patterns of aminoglycoside resistance in the Gram-negative bacteria isolates collected from pediatric and general hospitals

The purpose of the current study was to evaluate the phenotypic and genotypic patterns of aminoglycoside resistance among the Gram-negative bacteria (GNB) isolates collected from pediatric and general hospitals in Iran. A total of 836 clinical isolates of GNB were collected from pediatric and general hospitals from January 2018 to the end of December 2019. The identification of bacterial isolates was performed by conventional biochemical tests. Susceptibility to aminoglycosides was evaluated by the disk diffusion method (DDM). The frequency of genes encoding aminoglycoside-modifying enzymes (AMEs) was screened by the PCR method via specific primers. Among all pediatric and general hospitals, the predominant GNB isolates were Acinetobacter spp. (n = 327) and Escherichia coli (n = 144). However, E. coli (n = 20/144; 13.9%) had the highest frequency in clinical samples collected from pediatrics. The DDM results showed that 64.3% of all GNB were resistant to all of the tested aminoglycoside agents. Acinetobacter spp. and Klebsiella pneumoniae with 93.6%, Pseudomonas aeruginosa with 93.4%, and Enterobacter spp. with 86.5% exhibited very high levels of resistance to gentamicin. Amikacin was the most effective antibiotic against E. coli isolates. In total, the results showed that the aac (6')-Ib gene with 59% had the highest frequency among genes encoding AMEs in GNB. The frequency of the surveyed aminoglycoside-modifying enzyme genes among all GNB was found as follows: aph (3')-VIe (48.7%), aadA15 (38.6%), aph (3')-Ia (31.3%), aph (3')-II (14.4%), and aph (6) (2.6%). The obtained data demonstrated that the phenotypic and genotypic aminoglycoside resistance among GNB was quite high and it is possible that the resistance genes may frequently spread among clinical isolates of GNB.

In Vitro Activity of Various Sulbactam Compounds and Piperacillin/Tazobactam against Clinical Isolates of Different Gram-Negative Bacteria.

To provide direction for clinical application and pharmaceutical exploitation, the in vitro activity of sulbactam compounds and PIP/TAZ 8 : 1 against clinical isolates of Gram-negative bacteria (GNB, n = 976) was evaluated according to Clinical and Laboratory Standards Institute (CLSI) 2019. By minimal inhibitory concentrations (MICs), the resistance rate of all GNB to AMP/SBT 2 : 1 (56.9-100%) was significantly higher than other drugs, except the resistance rate of Acinetobacter baumannii (Aba, n = 204) to piperacillin/tazobactam (PIP/TAZ 8 : 1, 78.4%) which was close to it (76.5%). Additionally, the resistance rate of Aba to other compounds except AMP/SBT 2 : 1 differed greatly, but that of Klebsiella pneumonia (Kpn, n = 205) varied rarely. In addition, Escherichia coli (Eco, n = 204) and Kpn demonstrated low and high resistance rates, respectively. Compared with cefoperazone/sulbactam (CPZ/SBT 2 : 1), PIP/TAZ 8 : 1 had advantage in anti-Eco (RR = 0.5and OR = 2.17) and anti-Kpn activity (RR = 0.88and OR = 1.27), while its activity against Pseudomonas aeruginosa (Pae: n = 194, RR = 0.91, and OR = 1.12), Aba (RR = 1.31 and OR = 0.41), and other Enterobacteriaceae (other Ebc: n = 169, RR = 1.40, and OR = 0.62) was not better than CPZ/SBT 2 : 1. Although it had advantage against Eco (RR = 0.60 and OR = 1.78), Pae (RR = 0.67 and OR = 1.63), and Aba (RR = 0.70 and OR = 2.05), the inhibition effect of piperacillin/sulbactam (PIP/SBT 2 : 1) against Kpn (RR = 0.94 and OR = 1.12) and other Ebc was just similar with CPZ/SBT 2 : 1 (RR = 0.93 and OR = 1.10). Furthermore, the anti-Eco (RR = 0.70 and OR = 1.50), anti-Kpn (RR = 0.89 and OR = 1.24), and anti-Pae (RR = 0.74 and OR = 1.46) activities of ceftazidime/sulbactam (CAZ/SBT 1 : 1) had a weak advantage, while its activity against Aba (RR = 0.94 and OR = 1.15) and other Ebc (RR = 0.79 and OR = 1.36) was just close to CPZ/SBT 2 : 1. Moreover, the inhibitory effect of PIP/SBT 1 : 1 against all tested clinical species was more active than CPZ/SBT 2 : 1, while that of CAZ/SBT 2 : 1 against all species of bacteria analyzed was weaker than the controls.

A Physical-Chemical Study of the Interference of Ceftriaxone Antibiotic with Copper Chloride Salt.

The nano-CuCl2.2H2O salt was prepared by the ball milling method. The association parameters for bulk and nano-CuCl2 salts in H2O are estimated at different temperatures using the conductivity method by applying the Fuoss–Shedlovsky equation and it was higher for nano-CuCl2 than bulk CuCl2 salt. The interaction between the cation (Cu2+) and ligand (ceftriaxone) in H2O was determined also by the conductometric method. Two stoichiometric complexes 1/2 and 1/1 (M/L) are estimated and follow the order Kf (1/1) > Kf (1 : 2) and ∆Gf (1/1) > ∆Gf (1/2) for (M : L) (in negative values) indicate the favorable of formation of (1/1) complex compared to the (1 : 2) complex. The Gibbs free energies change was increased in negative signs with increasing the temperature. The antimicrobial activities of CFT, bulk Cu-CFT complex, and nano-Cu-CFT complex were studied on LB agar by the disc diffusion technique against clinical isolates of gram-negative bacteria (Klebsiella pneumonia and Pseudomonas aeruginosa) and Fungi (Candida albicans). It was observed that (CFT) has a higher zone of inhibition and antibacterial activity than that of bulk and nano-Cu-CFT complexes in Klebsiella pneumonia and Pseudomonas aeruginosa (gram-negative bacteria). The nano-Cu-CFT complex showed a higher clear zone of inhibition and antifungal activity against candida than bulk Cu-CFT complex while the absence of the inhibition zone in CFT, so nano-Cu-CFT complex, can be used as an antifungal drug.

A Multi-Point Surveillance for Antimicrobial Resistance Profiles among Clinical Isolates of Gram-Negative Bacteria Recovered from Major Ha'il Hospitals, Saudi Arabia.

The devastating nosocomial resistance is an on-going global concern. Surveillance of resistance is crucial for efficient patient care. This study was aimed to conduct a surveillance in four major Ha’il Hospitals from September to December 2020. Using a multipoint program, records of 621 non-duplicate Gram-negative cultures were tested across 21 drugs belonging to different categories. Major species were Klebsiella pneumoniae (n = 187, 30%), E. coli (n = 151, 24.5%), Pseudomonas aeruginosa, (n = 84, 13.6%), Acinetobacter baumannii (n = 82, 13.3%), and Proteus mirabilis (n = 46, 7%). Based on recent resistance classifications, A. baumanni, P. aeruginosa, and enteric bacteria were defined as pan-resistant, extremely resistant, and multi-drug resistant, respectively. A. baumannii (35%) and K. pneumoniae (23%) dominated among coinfections in SARS-CoV2 patients. The “other Gram-negative bacteria” (n = 77, 12.5%) from diverse sources showed unique species-specific resistance patterns, while sharing a common Gram-negative resistance profile. Among these, Providencia stuartii was reported for the first time in Ha’il. In addition, specimen source, age, and gender differences played significant roles in susceptibility. Overall infection rates were 30% in ICU, 17.5% in medical wards, and 13.5% in COVID-19 zones, mostly in male (59%) senior (54%) patients. In ICU, infections were caused by P. mirabilis (52%), A. baumannii (49%), P. aeruginosa (41%), K. pneumoniae (24%), and E. coli (21%), and most of the respiratory infections were caused by carbapenem-resistant A. baumannii and K. pneumoniae and UTI by K. pneumoniae and E. coli. While impressive IC, hospital performances, and alternative treatment options still exist, the spread of resistant Gram-negative bacteria is concerning especially in geriatric patients. The high selective SARS-CoV2 coinfection by A. baumannii and K. pneumoniae, unlike the low global rates, warrants further vertical studies. Attributes of resistances are multifactorial in Saudi Arabia because of its global partnership as the largest economic and pilgrimage hub with close social and cultural ties in the region, especially during conflicts and political unrests. However, introduction of advanced inter-laboratory networks for genome-based surveillances is expected to reduce nosocomial resistances.

Formation <i>in vitro</i> of colistin resistance in carbapenem-resistant Gram-negative bacteria and its biological cost

Introduction. The spread of resistance to carbapenems among gram-negative bacteria have led to an increase in the consumption of polymyxins and the emergence of certain strains resistant to them. Polymyxin resistance is mainly associated with mutations in chromosomal genes. The development of mutational resistance to antibiotics can lead to a decrease in the viability of bacteria; which is manifested by an increase in the duration of the cell cycle; a decrease in virulence and competitive fitness.The purpose of the study was to assess in vitro the intensity of the formation of colistin resistance in carbapenemresistant clinical isolates of gram-negative bacteria; the stability of the formed emerged resistance and its biological cost.Materials and methods. For 46 strains of Klebsiella pneumoniae; 77 strains of Pseudomonas aeruginosa and 42 strains of Acinetobacter baumannii; real time polymerase chain reaction (PCR) was used to detect the genes of carbapenemases; the minimum inhibitory concentrations (MIC) of meropenem and colistin were determined by broth microdilution method. The selection of resistant subpopulations on Muller–Hinton agar with the addition of 16 mg/l colistin was carried out. For colistin-resistant mutants and their isogenic sensitive strains; the kinetic parameters of growth in broth culture were determined. Incubation and result recording were performed on an Infinite M200 microplate reader for 18.5 hours at 35°C with measurement of light scatter in the wells every 15 minutes.Results. The production of carbapenemases MBL VIM in P. aeruginosa; MBL NDM; KPC and OXA-48 in K. pneumoniae; OXA-23 and OXA-40 in A. baumannii was observed. All strains were sensitive to colistin (MIC varied from 0.062 to 2 mg/l). The colony growth on a selective medium with16 mg/l colistin was observed for 97.8% of K. pneumoniae strains; 16.9% of P. aeruginosa strains; and 61.9% of A. baumannii strains. The mutational nature of colistin resistance was confirmed for 21.7% of K. pneumoniae strains. For colistin-resistant mutants of K. pneumoniae; a significant increase in the duration of the lag phase (Tlag) was observed: 225.6 ± 7.037 min in the wild-type susceptible strains and 245.5 ± 8.726 in resistant mutants; p = 0.037. The indicators of the doubling time of the number of microbial cells in the exponential growth phase (Tdoubling) and the area under the bacterial growth curve did not differ significantly.Conclusion. A high frequency of formation of colistin resistance in vitro in carbapenemase-producing strains of K. pneumoniae was observed. The absence of significant changes in the kinetics of microbial growth in resistant strains makes it possible to predict the further spread of mutational resistance to colistin; as well as its preservation in microbial populations of K. pneumoniae even in the case of limiting the use of this antibiotic.

Synergistic Antibacterial and Antibiofilm Activity of the MreB Inhibitor A22 Hydrochloride in Combination with Conventional Antibiotics against Pseudomonas aeruginosa and Escherichia coli Clinical Isolates.

In the era of antibiotic resistance, the bacterial cytoskeletal protein MreB is presented as a potential target for the development of novel antimicrobials. Combined treatments of clinical antibiotics with anti-MreB compounds may be promising candidates in combating the resistance crisis, but also in preserving the potency of many conventional drugs. This study aimed to evaluate the synergistic antibacterial and antibiofilm activities of the MreB inhibitor A22 hydrochloride in combination with various antibiotics. The minimum inhibitory concentration (MIC) values of the individual compounds were determined by the broth microdilution method against 66 clinical isolates of Gram-negative bacteria. Synergy was assessed by the checkerboard assay. The fractional inhibitory concentration index was calculated for each of the A22-antibiotic combination. Bactericidal activity of the combinations was evaluated by time-kill curve assays. The antibiofilm activity of the most synergistic combinations was determined by crystal violet stain, methyl thiazol tetrazolium assay, and confocal laser scanning microscopy analysis. The combined cytotoxic and hemolytic activity was also evaluated toward human cells. According to our results, Pseudomonas aeruginosa and Escherichia coli isolates were resistant to conventional antibiotics to varying degrees. A22 inhibited the bacterial growth in a dose-dependent manner with MIC values ranging between 2 and 64 μg/mL. In combination studies, synergism occurred most frequently with A22-ceftazidime and A22-meropemen against Pseudomonas aeruginosa and A22-cefoxitin and A22-azithromycin against Escherichia coli. No antagonism was observed. In time-kill studies, synergism was observed with all expected combinations. Synergistic combinations even at the lowest tested concentrations were able to inhibit biofilm formation and eradicate mature biofilms in both strains. Cytotoxic and hemolytic effects of the same combinations toward human cells were not observed. The findings of the present study support previous research regarding the use of MreB as a novel antibiotic target. The obtained data expand the existing knowledge about the antimicrobial and antibiofilm activity of the A22 inhibitor, and they indicate that A22 can serve as a leading compound for studying potential synergism between MreB inhibitors and antibiotics in the future.

Comparative analysis of bactericidal properties of synthetic peptides from the active center of GM-CSF - ZP2 against different gram-negative bacteria

Objective of this study was to carry out comparative analysis of bactericidal activity of synthetic peptide ZP2 (SP ZP2) against museum strains and clinical isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Materials and methods. We used museum strains of E. coli (ATCC 25922) and P. aeruginosa ATCC 27853, as well as 104 clinical isolates of Enterobacteriaceae, including E. coli (n = 22) and K. pneumoniae (n = 82), and 98 clinical isolates of non-fermenting Gramnegative bacteria, including P. aeruginosa (n = 43) and A. baumannii (n = 55), isolated from patients with various gynecological and surgical infections. Bactericidal activity of SP ZP2 (final concentration 10 μg/ml) against the microorganisms was assessed by difference in optical density (OD) for experimental and control broth cultures after 20 min of contact of bacterial suspensions (5 x 108 CFU/ml) with SP ZP2 (in control — with distilled water), adding meat-peptone broth and 4-hour incubation at 37 °C. The effect of SP ZP2 was expressed by the Bactericidal Activity Index (BAI, %). Results. Using the in vitro assays, we have found that SP ZP2 had a pronounced bactericidal effect on both the reference strains of E. coli and P. aeruginosa, and majority (95.5-98.2%) of the studied clinical isolates of Gram-negative bacteria, regardless of their species. With regard of the average BAI values, the tested bacterial species may be ranked by increasing their sensitivity to the synthetic ZP2 peptide as follows: P. aeruginosa (74.0±2.3%) — E. coli (77.6±3.5%) — K. pneumoniae (82.8±1.6%) — A. baumannii (84.3±1.7%). In addition, significant intraspecific variability of clinical isolates of Gram-negative bacteria was revealed for their sensitivity to bactericidal effect of SP ZP2. Conclusion. The synthetic ZP2 peptide is able to inhibit growth of Gram-negative bacteria, as well as exerts a bactericidal effect, thus considering it as a promising candidate for development of new effective drugs with combined immunobiological properties for combatting infectious and inflammatory conditions caused by the indicated microorganisms which show common resistance to a wide range of antimicrobial drugs used in clinical practice.

Evaluation of Vitek®2 performance for colistin susceptibility testing for Gram-negative isolates.

BackgroundThe emerging resistance to the last-resort antimicrobial colistin is being reported globally. Underestimation of the burden of colistin resistance and misinterpretation of colistin susceptibility test results, using suboptimal testing methods, may be causing unexplained treatment failures and even mortality among critically ill patients. Thus, this study was conducted at an apex trauma centre to assess the performance of Vitek®2 for colistin susceptibility testing.MethodsA total of 910 clinical isolates of Gram-negative bacteria (GNB), including Enterobacterales, Acinetobacter baumannii and Pseudomonas aeruginosa, were tested and analysed for colistin resistance using Vitek®2. Broth microdilution (BMD) was taken as the reference method. The essential (EA) and categorical (CA) agreements and very major error (VME) and major error (ME) rates were calculated. An MIC correlation was taken to be positive with EA ≥ 90%, CA ≥ 90%, VME ≤ 1.5% and ME ≤ 3.0% rates. Spearman’s coefficient was calculated and P < 0.05 was considered statistically significant.ResultsA total of 64% of isolates were MDR. Overall, 196 (21.5%) and 110 (12%) of isolates were resistant to colistin by BMD and Vitek®2, respectively. The automated Vitek®2 method failed to detect the resistance in up to 48.5% of GNB tested. When comparing Vitek®2 colistin interpretive results with reference BMD for all 910 isolates, the CA was 88% (798/910) with 10% (95/910) VMEs and 1% (9/910) MEs.ConclusionsThe Vitek®2 method for colistin susceptibility testing, still in use in some settings; is a suboptimal and unreliable method.

"Clicking" an Ionic Liquid to a Potent Antimicrobial Peptide: On the Route towards Improved Stability.

A covalent conjugate between an antibacterial ionic liquid and an antimicrobial peptide was produced via “click” chemistry, and found to retain the parent peptide’s activity against multidrug-resistant clinical isolates of Gram-negative bacteria, and antibiofilm action on a resistant clinical isolate of Klebsiella pneumoniae, while exhibiting much improved stability towards tyrosinase-mediated modifications. This unprecedented communication is a prelude for the promise held by ionic liquids -based approaches as tools to improve the action of bioactive peptides.

Spectrum of Aminoglycoside Modifying Enzymes in Gram-Negative Bacteria Causing Human Infections.

Introduction Aminoglycosides are formidable broad-spectrum antibiotics used in clinical settings; woefully their usage has been reduced by the emergence and distribution of resistance mainly due to aminoglycoside modifying enzymes (AME). Purpose This study was performed to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of gram-negative bacteria. This study also aimed to detect the presence of AMEs that are prevalent in gram-positive bacteria, among gram negatives. Materials and Methods A total number of 386 clinical isolates were included in this study. Polymerase chain reaction revealed the prevalence rate of AMEs screened [aac(6′)-lb, aac(3′)-I, aac(3′)-II, aac(3′)-VI, ant(2′)-I, ant(4′)-IIb, aac(3′)-III, aac(3′)-IV, aph(2′)-Ib, aph(2′)-Ic, aph(2′)-Id, aac (6′)-Ie- aph(2′)-Ia, and aph(3′)-IIIa]. Conjugation experiment was performed for the clinical isolates which harbored any one of the AME which was prevalent in gram-positive bacteria [aph(3′)-IIIa, aac(6′)-Ie-aph(2′)-Ia]. Results aac(6′)-lb is the most prevalent AME, followed by aac(3′)-I, aph(3′)-VI, aac(3′)-VI, and aac(3′)-II. The AMEs such as ant (2′)-I, ant(4′)-IIb, aac(3′)-III, aac(3′)-IV, aph(2′)-Ib, aph(2′)-Ic, and aph(2′)-Id were not established in our study isolates. The rate of prevalence of aph(3′)-IIIa, aac(6′)-Ie-aph(2′)-Ia—the AMEs encountered in gram-positive and their co-existence was 19.68% and the conjugation experiment revealed their transfer via plasmids. Conclusion This is the first report from India revealing the presence and prevalence of AMEs which are often encountered among gram-positive bacteria in gram negatives and their presence on conjugative plasmids.

Antimicrobial activity of POL7306 tested against clinical isolates of Gram-negative bacteria collected worldwide.

POL7306 belongs to a new class of peptidomimetic outer-membrane-protein-targeting antibiotics with a novel mechanism of action. POL7306 is in development for the treatment of infections caused by antimicrobial-resistant Gram-negative bacteria and has demonstrated low cytotoxicity and nephrotoxicity. A total of 891 isolates were collected by the SENTRY Antimicrobial Surveillance Program from 134 medical centres in Europe (n = 424; 41 centres in 18 nations), the USA (n = 411 isolates from 67 centres), the Asia-Pacific region (n = 24; 15 centres in 6 nations) and Latin America (n = 32; 11 centres in 9 nations) and included 558 Enterobacterales, 310 non-fermenters and 23 fastidious organisms. Susceptibility testing was performed using the reference broth microdilution method and the medium was supplemented with 0.002% polysorbate-80 for testing POL7306. Resistant subsets were characterized by WGS. POL7306 demonstrated potent in vitro activity against Enterobacterales [including carbapenem-resistant (MIC50/90, 0.06/0.25 mg/L), ESBL-producing (MIC50/90, 0.06/0.12 mg/L), KPC-producing (MIC50/90, 0.12/0.25 mg/L), MBL-producing (MIC50/90, 0.06/0.25 mg/L), colistin-non-susceptible, mcr-negative (MIC50/90, 0.5/2 mg/L) and mcr-positive (MIC50/90, 0.12/0.25 mg/L) Enterobacterales], Pseudomonas aeruginosa (MIC50/90, 0.25/0.25 mg/L), Acinetobacter baumannii (MIC50/90, 0.06/0.12 mg/L) and Stenotrophomonas maltophilia (MIC50/90, 0.06/0.25 mg/L). POL7306 demonstrated potent activity against a large collection of Gram-negative organisms collected worldwide that included colistin-resistant, XDR and ESBL- and carbapenemase-producing isolates for which there are currently limited treatment options.