Clinical Isolates Of Bacteria Research Articles

Prevalence of Bacterial Biofilms in Patients of Chronic Rhino Sinusitis (CRS) with or Without Nasal Polyposis

Background: Objective. There is growing evidence that bacterial biofilms may play a role in certain cases of recalcitrant CRS that do not respond to traditional medical and surgical therapies. Aim: The aim of this study was to determine the prevalence of biofilm-forming bacteria in clinical isolates of CRS patients with or without nasal polyposis. Methods: A Prospective observational study conducted in the Department of ENT & HNS GMC Srinagar and Department of Microbiology, from September 2018 to August 2020. 60 patients were enrolled in this study who reported to ENT & HNS OPD of Govt. Medical College Srinagar and diagnosed with CRS according to “clinical practice guidelines” 2015 criteria, and were willing to participate in the study. Samples were harvested in the OPD and/or intraoperatively for microscopic examination to determine biofilm presence. Statistical analysis was performed. For all statistical tests, P = .05 was considered significant. Results: In this study on 60 patients Biofilms were present in 32 (53.3%) patients whereas in 28 (46.7%) patients Biofilms were absent (includes 14 culture positive and 14 culture negative patients). Biofilm formation was detected by three different phenotypic methods. Biofilm detection rate were highest by Tissue Culture Plate (TCP) method (56.3%) followed by Tube method (TM) as (46.9%) and Congo Red Agar (CRA) method as (43.8%). Conclusion: This study further elucidates the prevalence of bacterial Biofilms in sinonasal mucosa of CRS patients as Biofilms existed in 53% of the patients under study.

Long-term Storage of Bacterial Isolates by Using Tryptic Soy Broth with 15% Glycerol in The Deep Freezer (-70 to -80 °C)

For different bacterial preservation techniques, there is no single method applicable for all bacteria. This study aimed to assess the viability of seven species/species groups of clinical bacteria isolates on the long-term storage (more than 5 years) by using Tryptic Soy Broth with 15% glycerol in the deep freezer (-70 to -80°C). A total 10,654 clinical bacteria isolates used as samples in this study. The isolates consisted of seven species/species groups (i.e. Escherichia coli, Campylobacter spp, Shigella spp, Vibrio spp, Salmonella spp, Aeromonas hydrophila, and Neisseria gonorhoeae). The isolates were collected from some previous studies and preserved in the Tryptic Soy Broth (TSB) with 15% glycerol and stored in the deep freezer (-70 to -80°C) for more than five years. The samples were revived on the suitable medium to evaluate the viability of bacteria. Identification conducted by microscopic examination, biochemical test, and latex agglutination. The study showed that the viability of Salmonella spp, Shigella spp, Aeromonas hydrophila and E. coli was 100%, while Campylobacter spp, Vibrio spp, and N. gonorhoeae were 66.7%, 66.4%, and 52.5% respectively. We concluded that viability of Salmonella spp, Shigella spp, A. hydrophila, and E. coli was optimum thus better than Campylobacter spp, Vibrio spp, and N. gonorhoeae for more than 5 years storage by using TSB with 15% glycerol in the deep freezer (-70 to -80 °C).

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.

Retraction Note: IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance.

Isoprenoids are vital for all organisms, in which they maintain membrane stability and support core functions such as respiration1. IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential for Gram-negative bacteria, mycobacteria and apicomplexans2,3. Its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), is not produced in metazoans, and in humans and other primates it activates cytotoxic Vγ9Vδ2 T cells at extremely low concentrations4–6. Here we describe a class of IspH inhibitors and refine their potency to nanomolar levels through structure-guided analogue design. After modification of these compounds into prodrugs for delivery into bacteria, we show that they kill clinical isolates of several multidrug-resistant bacteria—including those from the genera Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus—yet are relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with these prodrugs resemble those after conditional IspH knockdown. Notably, these prodrugs also induce the expansion and activation of human Vγ9Vδ2 T cells in a humanized mouse model of bacterial infection. The prodrugs we describe here synergize the direct killing of bacteria with a simultaneous rapid immune response by cytotoxic γδ T cells, which may limit the increase of antibiotic-resistant bacterial populations. A class of compounds with a dual mechanism of action—direct targeting of IspH and stimulation of cytotoxic γδ T cells to enhance pathogen clearance—are active against multidrug-resistant bacteria.

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.

Amphipathic Barbiturates as Mimics of Antimicrobial Peptides and the Marine Natural Products Eusynstyelamides with Activity against Multi-resistant Clinical Isolates.

We report a series of synthetic cationic amphipathic barbiturates inspired by the pharmacophore model of small antimicrobial peptides (AMPs) and the marine antimicrobials eusynstyelamides. These N,N'-dialkylated-5,5-disubstituted barbiturates consist of an achiral barbiturate scaffold with two cationic groups and two lipophilic side chains. Minimum inhibitory concentrations of 2-8 μg/mL were achieved against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum β-lactamase-carbapenemase production. The guanidine barbiturate 7e (3,5-di-Br) demonstrated promising in vivo antibiotic efficacy in mice infected with clinical isolates of Escherichia coli and Klebsiella pneumoniae using a neutropenic peritonitis model. Mode of action studies showed a strong membrane disrupting effect and was supported by nuclear magnetic resonance and molecular dynamics simulations. The results express how the pharmacophore model of small AMPs and the structure of the marine eusynstyelamides can be used to design highly potent lead peptidomimetics against multi-resistant bacteria.

PHYTOCHEMICAL ANALYSIS AND EVALUATION OF ANTIBACTERIAL ACTIVITY OF TOKAR SHA AGAINST ENTERIC BACTERIA

The antibacterial activity of Tokar sha; a local traditional medication widely used by many people in North-west zone of Nigeria especially Sokoto, Kebbi and Zamfara against enteric infections were examined against some clinical isolates of pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Bacillus cereus and Salmonella typhi) using agar well diffusion method. The pattern of inhibition varied with the tokar sha concentrations and the organisms tested. The tokar sha was more effective on E. coli with a maximum zone of growth inhibition of 25mm at 35mg/ml followed by B. cereus (20mm). However, S. aureus and S. typhi were resistant to tokar sha at all concentrations tested. The minimum inhibitory concentrations (MIC) were found to be 35mg/ml for both E. coli and B. cereus. The antibacterial activities exhibited by tokar sha in this study could be attributed to the presence of its constituents which signifies the potential of the tokar sha as a therapeutic agent. These findings may justify the ethnomedicinal use of tokar sha as an antibacterial agent against enterobacteria

Survey on antimicrobial resistance and virulence-related genes in Enterococcus faecium and Enterococcus faecalis collected from hospital environment in the north of Iran

Environmental contaminants in hospital settings play a major role in the spreading of pathogenic bacteria during outbreaks. The aim of this study was to evaluate the antibiotic resistance and virulence-related genes among Enterococcus strains isolated from hospital environment. Throughout this cross-sectional work, 388 samples were collected from hospital environment. Totally, 88 Enterococcus positive strains were characterized with diagnostic and confirmatory tests. Antibiogram was done according to the CLSI guideline. Genes encoding antibiotic resistance and virulence factors were detected by multiplex PCR. Out of 90 tested isolates, 53.33% and 46.6% of them were identified as E. faecium and E. faecalis, respectively. Also, multidrug resistance phenotype observed in 66.6% and 37.5% of E. faecalis and E. faecium strains, respectively. Distribution of resistance genes was as follows: ermB (66.6%), tetM and tetL (60%), aac(6′)-Ie-aph(2″)-Ia (28.8%), aph (3′)-IIIa (26.6%), and vanA, vanB (8.8%). Moreover, 97.7% and 17.7% of the isolates, carried virulence factors of esp and ace, respectively. This report showed that the environmental Enterococcus are the major sources of resistance genes that can transfer them to the clinical isolates of bacteria in hospital settings.

Prevalensi Gen OXA-23 pada Isolat Klinis Bakter Acinetobacter baumanii di RSUP Dr. M. Djamil Padang

Acinetobacter baumannii is such a big challenge in this modern era because of their multi-drug resistance (MDR) through their resistance mechanism, namely the production of beta lactamase enzymes, one of them is oxacillinases. Several studies have revealed that the OXA-23 gene has an important role in the production of the beta lactamase enzyme so that these bacteria are resistant to antibiotics, especially carbapenem. This study was conducted to determine the description of the resistance of the Acinetobacter baumannii by looking at the prevalence of the OXA-23 gene in clinical isolates of these bacteria at RSUP Dr. M. Djamil Padang.
 This study was a descriptive observational study and used a cross-sectional design using polymerase chain reactions (PCR) and electrophoresis techniques. The samples were clinical isolates of Acinetobacter baumannii bacteria stored in the Microbiology Laboratory of the Faculty of Medicine, Andalas University with inclusion and exclusion criteria.
 Of the 150 clinical isolates of Acinetobacter baumannii studied, most of the bacteria were still sensitive to the antibiotics of Amikacin (84%), Tigecycline (78.76%), and Trimethoprim / Sulfamethoxazole (76.77%). Meanwhile, all of the Acinetobacter baumannii isolates were resistant to cefazolin. In PCR and electrophoresis tests, 50 clinical isolates were positive for the OXA-23 gene. 46 of them were carbapenem resistant, while the other 4 isolates were sensitive to carbapenem. From these results it can be concluded that the prevalence of the OXA-23 gene in Acinetobacter baumannii isolates in RSUP Dr. M. Djamil Padang was 85.18% for carbapenem resistant isolates and 4.30% for carbapenem sensitive isolates.
 Keywords : Acinetobacter baumannii, carbapenem, OXA-23 gene

New cyclolignans of Larrea tridentata and their antibacterial and cytotoxic activities

• Larrea tridentata is a source of new antibacterial and cytotoxic cyclolignans. • Methylation of cyclolignans can enhance the antibacterial and cytotoxic activity. • Drug-resistant gram-positive bacteria is susceptible to cyclolignans antibacterial activity. • Methylated cyclolignans shows antimycobacterial activity against drug-resistant Mycobacterium tuberculosis . From the chloroform extract of Larrea tridentata (Ses. et Moc ex DC.) Coville two new cyclolignans (1 and 2) and six known compounds (3–8) were isolated and characterized. The new compounds were elucidated as 4,4’-dihydroxy-3-methoxy-6,7’-cyclolignan (1) and 3,4-dihydroxy-3’,4’-dimethoxy-6,7’-cyclolignan (2). Compound 1 was methylated to obtain the semi-synthetic derivatives 1a, 1b and, 1c. The structural elucidation of compounds was performed by analysis of 1D and 2D NMR spectral data, HRMS and, X-ray diffraction (compound 1). Antibacterial and cytotoxic activity of compounds was determined against nine clinical isolates of drug-resistant bacteria and two strains of M. tuberculosis (drug-sensitive H37Rv and drug-resistant G122) as well as against three cancer cell lines. Results showed the methylated derivative 1a as the most active antibacterial compound, displaying the best activities against gram-positive bacteria and M. tuberculosis with a minimal inhibitory concentration (MIC) in a range of 6.25–12.5 μg/mL and 12.5−25 μg/mL respectively, having the same activity as the control drug levofloxacin against methicillin-resistant Staphylococcus aureus (MIC 6.25 μg/mL) and linezolid-resistant Staphylococcus epidermidis (MIC 12.5 μg/mL). Cyclolignan 5 was two-fold more active towards (LR) S. epidermidis. Tested compounds were devoid of cytotoxic activity.

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.

Analysis of Selection Methods to Develop Novel Phage Therapy Cocktails Against Antimicrobial Resistant Clinical Isolates of Bacteria.

Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis are E. coli and Klebsiella along with Enterobacter species. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as “critical” for research. Bacteriophages (phages), as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains. There is currently no “gold standard” for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producing E. coli and Klebsiella largely isolated from patients in United Kingdom hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species. A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay (PKA) and the biofilm assay. The final ESBL cocktail of six phages could effectively kill 23/38 strains (61%), for Klebsiella 13/19 (68%) and for E. coli 10/19 (53%) based on the PKA data. The ESBL E. coli collection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that PKAs are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows “phage synergy” to inform cocktail design.

The effect of EDTA in combination with some antibiotics against clinical isolates of gram negative bacteria in Mansoura, Egypt

Extensive use of antibiotics in clinical practice has been associated with increasing frequency of resistant microorganisms. So new strategy is needed to treat the resistant pathogens. Hence this study was conducted to determine the effect of Ethylenediaminetetraacetic acid (EDTA) in increasing the inhibition effect of some antibiotics on multi-drug resistant (MDR) gram-negative bacteria. For this purpose, 40 E. coli isolates, 40 K. pneumoniae isolates and 50 P. aeruginosa isolates were collected from different University's hospitals in Mansoura, Egypt. Antibacterial susceptibility pattern against 9 different antimicrobials were studied by disc diffusion method. Also the effect of two sub-inhibitory concentrations of EDTA (1 and 2 mM) on the inhibition zones of antibiotic discs against the highly multidrug resistant (MDR) isolates was determined. Checkerboard method was used for testing the activity of gentamicin/EDTA and cefotaxime/EDTA combinations on the highly MDR isolates. Additionally, the effect of EDTA on the expression of efflux pump genes was tested by real time-PCR. Most of the clinical isolates were found to be resistant to the tested antibiotics except imipenem and high prevalence of MDR isolates was recorded. 34 isolates were selected as those showed the highest multi-drug resistance and were tested to specify their MIC for EDTA as EDTA showed strong antibacterial activity with MIC ranging 4–8 mM. The addition of sub-MIC of EDTA (1or 2 mM) to the agar plate resulted in changing the 11 tested E. coli isolates from resistant to sensitive to ceftazidime, gentamicin, rifampin, ampicillin, erythromycin and vancomycin, the tested K. pneumoniae isolates were turned also from resistant to sensitive to gentamicin and ceftazidime, additionally the tested P. aeruginosa isolates became sensitive to gentamicin, ceftazidime and ciprofloxacin. Indifference to additive activity was observed for tested combinations and MIC value of cefotaxime or gentamicin in combination with EDTA was less than antibiotic alone in the most tested isolates. Moreover, significant reduction (P < 0.01) in the expression of all tested efflux pump genes in treated E. coli, K. pneumoniae and P. aeruginosa isolates with EDTA compared to untreated isolates was observed. In conclusion, these results suggest that the combination of antibiotic especially gentamicin with EDTA may be fruitful for management of resistant gram negative infections.

Coumarin-1,2,3-triazole Hybrid Molecules: An Emerging Scaffold for Combating Drug Resistance.

Undoubtedly, antibiotics have saved billions of lives, but lack of novel antibiotics, development of resistance mechanisms in almost all clinical isolates of bacteria, and recurrent infections caused by persistent bacteria hamper the successful treatment of the infections. Due to the widespread emergence of resistance, even the new families of anti-microbial agents have a short life expectancy. Drugs acting on a single target often lead to drug resistance and are associated with various side effects. For overcoming this problem, either multidrug therapy, or a single drug acting on multiple targets may be used. The latter is called 'hybrid molecules,' which are formed by clubbing two biologically active pharmacophores together, with or without an appropriate linker. In this rapidly evolving era, the development of natural product-based hybrid molecules may be a super-alternative to multidrug therapy, for combating drug resistance caused by various bacterial and fungal strains. Coumarins (benzopyran-2-one) are one of the earliest reported plant secondary metabolites having a clinically proven diverse range of pharmacological properties. On the other hand, 1,2,3-triazole is a common pharmacophore in many drugs responsible for polar interactions, improving the solubility and binding affinity to biomolecular targets. In this review, we discuss recent advances in Coumarin-1,2,3-triazole hybrids as potential anti-bacterial agents, aiming to provide a useful platform for the exploration of new leads with a broader spectrum, more effectiveness and less toxicity with multiple modes of action for the development of cost-effective and safer drugs in the future.

Detection of diverse carbapenem and multidrug resistance genes and high-risk strain types among carbapenem non-susceptible clinical isolates of target gram-negative bacteria in Kenya.

Carbapenem-resistant gram-negative bacteria are an increasingly significant clinical threat globally. This risk may be underestimated in Kenya as only four carbapenemase genes in three bacterial species have been described. The study aimed to understand the antibiotic resistance profiles, genes, sequence types, and distribution of carbapenem-resistant gram-negative bacteria from patients in six hospitals across five Kenyan counties by bacterial culture, antibiotic susceptibility testing, and whole-genome sequence analysis. Forty-eight, non-duplicate, carbapenem non-susceptible, clinical isolates were identified across the five counties (predominantly in Nairobi and Kisii): twenty-seven Acinetobacter baumannii, fourteen Pseudomonas aeruginosa, three Escherichia coli, two Enterobacter cloacae, and two Klebsiella pneumoniae. All isolates were non-susceptible to β-lactam drugs with variable susceptibility to tigecycline (66%), minocycline (52.9%), tetracycline (29.4%), and levofloxacin (22.9%). Thirteen P. aeruginosa isolates were resistant to all antibiotics tested. Eleven carbapenemase genes were identified: blaNDM-1, blaOXA-23, -58, -66, -69, and -91 in A. baumannii (STs 1, 2, 164 and a novel ST1475), blaNDM-1 in E. cloacae (STs 25,182), blaNDM-1, blaVIM-1and -6, blaOXA-50 in P. aeruginosa (STs 316, 357, 654, and1203), blaOXA-181, blaNDM-1 in K. pneumoniae (STs 147 and 219), and blaNDM-5 in E. coli (ST164). Five A. baumannii isolates had two carbapenemases, blaNDM-1, and either blaOXA-23 (4) or blaOXA-58 (1). AmpC genes were detected in A. baumannii (blaADC-25), E. cloacae (blaDHA-1 and blaACT-6, 16), and K. pneumoniae (blaCMY). Significant multiple-drug resistant genes were the pan-aminoglycoside resistance16srRNA methyltransferase armA, rmtB, rmtC, and rmtF genes. This study is the first to report blaOXA-420, -58, -181, VIM-6, and blaNDM-5 in Kenyan isolates. High-risk STs of A. baumannii (ST1475, ST2), E. cloacae ST182, K. pneumoniae ST147, P. aeruginosa (ST357, 654), and E. coli ST167, ST648 were identified which present considerable therapeutic danger. The study recommends urgent carbapenem use regulation and containment of high-risk carbapenem-resistant bacteria.

A Systematic Review of Intracellular Microorganisms within Acanthamoeba to Understand Potential Impact for Infection.

Acanthamoeba, an opportunistic pathogen is known to cause an infection of the cornea, central nervous system, and skin. Acanthamoeba feeds different microorganisms, including potentially pathogenic prokaryotes; some of microbes have developed ways of surviving intracellularly and this may mean that Acanthamoeba acts as incubator of important pathogens. A systematic review of the literature was performed in order to capture a comprehensive picture of the variety of microbial species identified within Acanthamoeba following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Forty-three studies met the inclusion criteria, 26 studies (60.5%) examined environmental samples, eight (18.6%) studies examined clinical specimens, and another nine (20.9%) studies analysed both types of samples. Polymerase chain reaction (PCR) followed by gene sequencing was the most common technique used to identify the intracellular microorganisms. Important pathogenic bacteria, such as E. coli, Mycobacterium spp. and P. aeruginosa, were observed in clinical isolates of Acanthamoeba, whereas Legionella, adenovirus, mimivirus, and unidentified bacteria (Candidatus) were often identified in environmental Acanthamoeba. Increasing resistance of Acanthamoeba associated intracellular pathogens to antimicrobials is an increased risk to public health. Molecular-based future studies are needed in order to assess the microbiome residing in Acanthamoeba, as a research on the hypotheses that intracellular microbes can affect the pathogenicity of Acanthamoeba infections.

Synthetic Molecular Evolution to Identify Hemocompatible Antimicrobial Peptides Effective Against Drug-Resistant, Biofilm-Forming Bacteria

Previously, we identified antimicrobial peptides (AMPs) that retain activity in the presence of eukaryotic cells by using a synthetic molecular evolution (SME) based approach. We demonstrated that variation in the sequence of antimicrobial peptides selected from primary screening of iterative peptide library can lead to identification of peptides that have superior antimicrobial activity, solubility, and lower cytotoxicity against red blood cells (RBCs) and mammalian cells. D-CONGA, a variant obtained by rational modification of the consensus sequence of peptides selected from stringent screening of iterative, second-generation antimicrobial peptide library is highly effective in vitro against all ESKAPE pathogens in the presence of RBCs and serum. The peptide also has anti-biofilm activity in vitro and shows potency against gram-negative and gram-positive drug-resistant bacteria and their biofilms in relevant infected wound model in mice. We next designed and synthesized 13 variants of D-CONGA and characterized them for their bactericidal activity in the presence and absence of RBCs. Their cytotoxicity against mammalian fibroblast, WI-38, and hemolysis in RBC were also measured. The best D-CONGA variant, D-CONGA-Q7, was then tested against 14 clinical isolates of drug-resistant bacteria. The anti-microbial activity of the new peptide was compared with D-CONGA and eight conventional antibiotics from four different classes and was found to be superior to all. Following this, we designed and synthesized a third-generation peptide library based on D-CONGA-Q7. The library members are being screened against gram-positive and gram-negative bacteria in the presence of RBCs. The selected peptides sterilize broad spectrum bacteria at submicromolar concentrations. Hence, we have shown that using rational design and SME, we can improve AMPs with each generation and discover evolved peptides that have increasingly potent and relevant activity against MDR bacteria, compared to conventional antibiotic or other known AMPs.

Liquid chromatography mass spectrometry-based proteomics of Escherichia coli single colony

The Escherichia coli proteome is the most extensively characterized and studied of all prokaryotic proteomes. Despite this, large scale bacterial proteomics experiments performed on E. coli cells grown in liquid cultures have failed to identify key virulence factors thought to be important determinants in establishing bacterial infections. It seems likely that many important determinants associated with virulence and host cell adhesion are exclusively expressed during growth in biofilms, which can be crudely mimicked on solid media. This method describes a simple workflow to characterize the unique proteome signature of individual, isolated single colonies, using E. coli K12 strain grown on solid media as a model system. The workflow thus provides a means to explore the proteomes of minimally passaged clinical isolates of bacteria grown on primary culture plates and to identify both unique and differentially expressed proteins contained therein.Value of the method:- Simple mass spectrometry-based proteomics workflow to characterise the proteome of single colony forming units- Enables exploration of the proteomes of minimally passaged clinical isolates from primary culture plates- Identification of virulence factors expressed in true or mimicked biofilms that may be missed in liquid culturesMethod name: E. coli single colony proteome analysis