Vitek System Research Articles

Pioneering study of Egyptian Neem and Jojoba extracts with molecular docking combat hospital multidrug resistant bacteria.

Hospital surfacesare often contaminatedwith multidrug-resistant pathogenic bacteria that cause healthcare-associated infections and lead to increased mortality and morbidity. Thereis a need for new alternative antibacterial agents to overcome antibiotic resistance. Azadirachta indica and Simmondsia chinensis have been found to possess antibacterial activity and medicinal value. The antibacterial activity of these plant extracts against clinical isolates was investigated using the agar disc diffusion method. These clinical isolates included E. coli, Pseudomonas aeruginosa, Acinetobacter spp., Klebsiella pneumoniae, Stenotrophomonas maltophilia, and methicillin-resistant Staphylococcus aureus (MRSA), which were identified by the vitek-2 system, and resistance genes of selected bacterial strains were identified by using the bioFire FilmArray test. The most potent extract of these plants was the ethanolic extract, where the inhibition percentage of ethanolic Jojoba and Neem extracts was 90.9% and 74.5%, respectively against all the tested pathogens. On the other hand, the methanolic extracts of Neem and Jojoba have different degrees of antibacterial activity against the tested pathogens. The phytochemical components of the most potent extracts (ethanolic extracts) were investigated by gas chromatography‒mass spectrometry (GC\MS), which revealed that the ethanolic extracts were enriched in phenolics, flavonoids, and sugars. FTIR analyses of the plant extracts confirmed the presence of alcoholic, carboxylic, and aldehydic moieties. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity of the ethanolic extracts of Neem and Jojoba increased in a dose-dependent manner, with average IC50 values of 98.17 ± 0.85, 4.95 ± 0.06, and 4.17 ± 0.04 mg/mL, respectively, for the ethanolic Neem extract, the ethanolic Jojoba extract, and ascorbic acid (standard). Furthermore, increased cytotoxicity was demonstrated in the HFB4 cell line in a dose-dependent manner. The average IC50s of the ethanolic Neem extract and the ethanolic Jojoba extract were 18.18 ± 0.15 and 76.16 ± 1.49 mg/mL, respectively. Moreover, the results for the antibiofilm activity of the ethanolic Neem extract showed that 99.5% of the biofilms formed at 25 mg/ml. Inaddition, 50 mg/ml of the ethanolic extract of Jojoba had a suppressive effect of 98.2%. The significant components Nonanoic acid (21.9405%) and Palmitic Acid (16.0869%) from Neem and pinitol from Jojoba (82.85%) were selected throughout the molecular docking investigation, by which the chosen constituents inhibited the crystal structure of penicillin-binding protein 4 (PBP4) from Staphylococcus aureus (PDB ID: 1TVF) and the crystal structure of the OXA-48 beta-lactamase (PDB ID: 7AUX) from K. pneumoniae. Overall, our study reveals the effectiveness of antimicrobial plant extracts as therapeutic solutions for antibiotic resistance in Egypt and worldwide with some modifications to decrease their cytotoxicity.

Inhibitory Effect of Shikimic Acid on Biofilm Formation and the Expression of Critical Biofilm-associated Genes in Streptococcus agalactiae

Background Streptococcus agalactiae is well-known for causing adverse pregnancy outcomes, such as stillbirths and miscarriages, as well as being a major cause of newborn sepsis and meningitis. Aims This study sought to investigate how shikimic acid influences the expression of genes involved in the synthesis of biofilms. Methods A total of 181 specimens were obtained from pregnant women. Twenty-two isolates were bacteriologically recognized as S. agalactiae from these specimens. They were gathered from hospitals in the Al-Anbar province. These twenty-two isolates were identified as Group B streptococcus (GBS) based on their cultural and microscopical properties, as well as automated (VITEK-2 system) and molecular identification based on the atr gene, which is an essential gene expressed in all S. agalactiae isolates. Results GBS produced varying amounts of biofilm (weak, moderate, and strong). Shikimic acid (SA) was tested for its antibacterial effect against biofilm. Shikimic acid was incubated at various doses, and its effects on biofilm growth and formation were evaluated by MTT and crystal violet assay. SA greatly reduced the GBS inhibitory effects on GBS biofilms in pregnant women. Furthermore, it demonstrated a potent initial cell attachment, but it had less inhibitory effects on biofilms that had already been developed on polystyrene surfaces after eight hours. However, the Checkerboard approach produced a synergistic interaction between erythromycin and shikimic acid. Polymerase Chain Reaction (PCR) was used to verify the presence of pilA and PilB genes in the GBS strain. We detected these genes by PCR. The results revealed that pilA was found only in three isolates (13.63%), but pilB was found in all isolates (22/22;100%). Conclusion Quantitative real-time PCR was used to determine the effect of shikimic acid on the expression of genes (sag1407 and sag1408), and the results showed down-regulation of the expression of biofilm synthesis genes sag1407 and sag1408 when treated with Sub-MIC of shikimic acid.

Epidemiological and genetic characteristics of clinical carbapenemase-producing Enterobacterales isolates from Batna hospitals in Algeria

BackgroundCarbapenemase-producing Enterobacterales isolates are associated with significant mortality and have emerged as a major problem in healthcare settings worldwide.ObjectiveOur aim was to investigate the epidemiological and genotypic characteristics of carbapenemase-positive Enterobacterales isolates from patients hospitalised in three hospitals in the city of Batna, Algeria.MethodsBetween 2016 and 2019, a total of 5,316 clinical isolates were obtained. The collected isolates were identified using the VITEK-2 system. Demographic and microbiological data were collected as well as the antimicrobial susceptibility testing, phenotypic and molecular characterisation of carbapenemase and mcr-1 genes were performed.ResultsOut of the 5,316 isolates, 201 were carbapenem-resistant Enterobacterales isolates and 179 of them (89.05%) were positive for the production of carbapenemase, of which Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae were the most common. The blaOXA−48−like gene alone was detected in 147 isolates (82.12%) moreover, the blaNDM gene was detected in ten isolates (5.59%). Dual and triple combinations of carbapenemase genes were also observed here for the first time in Algeria: blaVIM and blaOXA−48−like; blaKPC, blaVIM and blaOXA−48−like; blaVIM and blaNDM; blaKPC, blaNDM and blaVIM; blaNDM and blaOXA−48−like genes. In addition, resistance to both colistin and carbapenem antibiotics was detected in eight isolates, however none of them was positive for the mcr-1 gene.ConclusionThis is the first study reporting the detection of carbapenemase genes in Klebsiella aerogenes, Enterobacter sakazakii, Raoultella ornithinolytica, Serratia ficaria, and Serratia marcescens and specific carbapenemase gene combinations in Algeria. The present study revealed that blaOXA−48−like were found to be the predominant carbapenemase genes in Batna hospitals.

Tracking Multidrug Resistance in Gram-Negative Bacteria in Alexandria, Egypt (2020-2023): An Integrated Analysis of Patient Data and Diagnostic Tools.

The rise in carbapenem-resistant Enterobacteriaceae (CRE) in Egypt, particularly in hospital settings, poses a significant public health challenge. This study aims to develop a combined epidemiological surveillance tool utilizing the Microreact online platform (version 269) and molecular microarray technology to track and analyze carbapenem-resistant Escherichia coli strains in Egypt. The objective is to integrate molecular diagnostics and real-time data visualization to better understand the spread and evolution of multidrug-resistant (MDR) bacteria. The study analyzed 43 E. coli isolates collected from Egyptian hospitals between 2020 and 2023. Nanopore sequencing and microarray analysis were used to identify carbapenemase genes and other resistance markers, whereas the VITEK2 system was employed for phenotypic antibiotic susceptibility testing. Microreact was used to visualize epidemiological data, mapping the geographic and temporal distribution of resistant strains. We found that 72.09% of the isolates, predominantly from pediatric patients, carried the blaNDM-5 gene, while other carbapenemase genes, including blaOXA-48 and blaVIM, were also detected. The microarray method demonstrated 92.9% diagnostic sensitivity and 87.7% diagnostic specificity compared to whole-genome sequencing. Phenotypic resistance correlated strongly with next-generation sequencing (NGS) genotypic data, achieving 95.6% sensitivity and 95.2% specificity. This method establishes the utility of combining microarray technology, NGS and real-time data visualization for the surveillance of carbapenem-resistant Enterobacteriaceae, especially E. coli. The high concordance between genotypic and phenotypic data underscores the potential of DNA microarrays as a cost-effective alternative to whole-genome sequencing, especially in resource-limited settings. This integrated approach can enhance public health responses to MDR bacteria in Egypt.

Qualitative and Quantitative Detection of Virulence-Associated Genes in Escherichia Coli Isolates from Women with Urinary Tract Infections

Escherichia coli is the most common type of Enterobacteriaceae family that causes urinary tract infections. This bacterium causes diseases due to its many virulence factors. During the study, (150) urine samples were collected for women with symptoms of UTIs aged between (20-50) years from 1/9/2021 to 1/4/2022. Forty-five isolates of E. coli were diagnosed using a biochemical test, a VITEK2 system, and molecular detection using traditional PCR. A molecular study was conducted on 25 of these isolates using conventional PCR and qRT-PCR to investigate the presence of genes included (16S rRNA, uidA, fimA and kpsMTII). The traditional PCR results showed the presence of both 16S rRNA and uidA gene in all 25 isolates of E. coli. In contrast, the results showed that 23 (92%) and 18 (72%) isolates possessed fimA and kpsMTII genes, respectively. Following the extraction of RNA from twenty-five pathogenic isolates of E. coli and ten non-pathogenic isolates, gene expression of fimA and kpsMTII was quantified using quantitative real-time reverse transcription PCR (qRT-PCR). The observed fold change indicates a potential overexpression of the KSPMII and FimA genes in uropathogenic E. coli. The expression of the kpsMTII gene showed the highest level of folding with an average of 22.89 in the case of uropathogenic E.coli isolates compared with non-pathogenic isolates (control) with an average of 4.11. While the expression of the fimA gene with the highest level of fold change with an average of 90.52 in the case of uropathogenic E.coli isolates compared with non-pathogenic isolates (control) with an average of 0.94, Significant differences were observed among the isolates, as indicated by P values less than 0.05. This finding demonstrated that qRT-PCR detects genes that were expressed in comparison with traditional PCR. Also, qRT-PCR is a more sensitive and accurate assay than conventional PCR for determining the quantitative prevalence of E. coli in urine samples.

A Rare Report of Carbapenem Resistant Vibrio fluvialis Isolated from a case of Walled off Necrosis: A Sequelae of Acute Necrotising Pancreatitis

Vibrio fluvialis (V. fluvialis) is commonly found in coastal environments and is an emerging pathogen encountered in diarrhoeal outbreaks and sporadic extraintestinal cases. V. fluvialis infections usually manifest as watery bloody diarrhoea, nausea, vomiting, gastroenteritis and peritonitis. In this case report, an unusual presentation of Multidrug Resistant (MDR) V. fluvialis along with Klebsiella pneumoniae co-infection from a case of Walled Off Necrosis (WON)- A complication of Acute Necrotising Pancreatitis (ANP) is discussed. The patient presented with the acute abdomen and had peripancreatic collection for which Ultrasound (USG) guided 12F pigtail drain placed over the right lower quadrant of the abdomen and conservatively managed. Routine cultures were done and the growth on Sheep Blood Agar (SBA) plate and MacConkey (MAC) agar plates were subjected for automated phenotypic identification by Matrix Assisted Laser Desorption Ionisation Time of Flight-Mass Spectrometry (MALDITOF-MS) and Vitek-2 systems and was identified as V. fluvialis. Also, molecular tests like 16S rRNA Polymerase Chain Reaction (PCR) followed by Sanger’s sequencing was done and the identification was further confirmed. Phenotypic RESIST-5 TRURAPID O.K.N.V.I. commercial lateral flow immunochromatographic assay for detection of clinically relevant and common carbapenemase genes like NDM, OXA-48, VIM, IMP and KPC was done which revealed this V. fluvialis isolate as New Delhi Metallo betalactamase (NDM) type of Carbapenemase producer. Patient was managed with levofloxacin and doxycycline and patient condition improved and was discharged.

Targeting nuc Gene for Detection of Staphylococcus aureus from Bovine Milk Samples of Assam, India and Phenotypic Identification of Antibiotic Resistance - A New Insight

Staphylococcus aureus is the leading cause of milk-borne disease in animals and humans worldwide, and it is often contaminated by enterotoxigenic and antimicrobial-resistant S. aureus strains. The current research work was intended to identify the prevalence of S. aureus from samples of bovine milk from various dairy farms and local vendors of Kamrup Metro District, Assam, India, by phenotypic and genotypic identification along with antibiotic resistance profiling. The conventional aseptic methods were implemented for S. aureus isolation from milk in Baird Parker Agar, supplemented with egg yolk and potassium tellurite. Further, the isolates confirmation was carried out using the automated VITEK system and amplification of the S. aureus specific nuc gene by PCR. Antibiotic susceptibility profiling for variety of 16 antibiotics was obtained through the conventional disc diffusion method. Eighty-five presumptive isolates with jet-black colonies with a white halo on Baird Parker Agar were selected. Thirty-eight isolates were eventually confirmed as S. aureus by the automated method and the detection of nuc gene. Antibiotic profiling revealed about 60.52% of the isolates to be multidrug resistant and 55.26a ± 0.01 mm resistant against Kanamycin. The statistical analysis data expressed correlation between Penicillin G and Ampicillin with 42.10b ± 0.01 mm and correlation among Tetracycline, Methicillin and Streptomycin with 10.52h ± 0.01 mm, respectively. Resistance against Kanamycin, Trimethoprim, Cloxacillin, and Nalidixic acid is concerning as such a resistant pattern has not been extensively reported in bovine milk samples in India, which could indicate the possible emergence of MDR S. aureus strains in the study area.

Molecular Identification of OXA Carbapenemase-Encoding Genes in Acinetobacter baumannii Isolated from Patients in Critical Care in Egypt.

Background: The emergence of carbapenem-resistant Acinetobacter baumannii (CRAB) in hospitals, particularly within critical care units, has garnered substantial global concern. CRAB commonly arises from the degradation by various ß-lactamases. Objective: We aimed to assess OXA-type carbapenemases in clinical isolates of A. baumannii obtained from an Egyptian tertiary care facility. Patients and Methods: This study examined 25 distinct A. baumannii strains collected from various clinical samples of patients in intensive care unit. Bacterial identification was conducted utilizing both traditional methods and the Vitek2 system. Antibiotic resistance profiles were assessed according to the European Committee on Antimicrobial Susceptibility Testing standards using the Vitek2 Compact automated system. Additionally, multiplex real-time polymerase chain reaction was used to identify the presence of blaOXA23, blaOXA24, blaOXA51, and blaOXA58 carbapenemase genes. Colistin susceptibility was assessed utilizing the broth microdilution method. Results: Carbapenem resistance was identified in 100% of the studied isolates. The blaOXA51 gene was detected in all A. baumannii strains. The gene blaOXA23 was identified in 22 strains (88%), whereas blaOXA24 and blaOXA58 were present in 15 strains (60%). All isolates, except one, co-harbored two or more OXA encoding genes. Colistin resistance was detected in 4 of 25 strains (16%). Conclusion: Our findings demonstrate the widespread distribution of CRAB isolates that co-harbor multiple carbapenemase-encoding genes. Molecular epidemiological studies and the surveillance of antibiotic resistance profiles may aid in identifying and tracing the origins of resistant bacteria, thereby limiting their spread.

Prevalence and antibiogram pattern of multidrug-resistant Staphylococcus aureus infections in individuals with sickle cell disease. A retrospective study, hematological and genetic analysis

The prevalence of Staphylococcus aureus in the circulation (bacteremia) of individuals with sickle cell disease (SCD) may provide a substantial risk for illness and death. Strains of S. aureus exhibit a wide variety of virulence traits, including the ability to generate various toxins and develop treatment resistance. MRSA is characterized by its resistance to a range of beta-lactam antibiotics. This study’s objectives included assessing the frequency of S. aureus infection in SCA patients, analyzing laboratory markers of the CBC test, examining the antibiotic susceptibility pattern, and investigating the mecA gene and heteroresistance, which could potentially link to a higher occurrence of S. aureus infection in SCA patients. The retrospective study was done to gather clinical data on individuals with sickle cell anemia (SCA) who had bloodstream infections. From 2017 to 2021, blood samples and data were gathered by King Saud University Medical City (KSUMC) in Riyadh, Saudi Arabia. The S. aureus strains were re-cultivation, Vitek system and PCR techniques were employed to ascertain the antibiotic resistance patterns. It was confirmed that 2406 patients with SCD had contracted an infection. A total of 138 (5.73 %) patients with SCD were diagnosed with bacteremia. “Of the 138 strains isolated, the MRSA strain revealed as the most prevailing, including 30 isolates (21.7 %), whereas non-MRSA strains constituted 17 isolates (12.3 %).” Males had a higher rate of infection compared to females. The results indicated that all the S. aureus isolates exhibited resistance to ampicillin, while the resistance to penicillin was 98 %. Furthermore, the results indicated that over 65 % of the isolates exhibited resistance to amoxicillin, amoxicillin, cefazolin, imipenem, and oxacillin. The mecA gene was identified in all S. aureus strains obtained from patients with SCD. The current study recognizes the PCR assay as a more dependable technique for assessing antibiotic resistances, in contrast to the Vitek method. A study found strong links between S. aureus infections in individuals with SCD and three different factors: hemoglobin (Hb) level, mean corpuscular volume (RDW), and white blood cell (WBC) count (p < 0.004, 0.005, and 0.02, respectively). The study revealed that 34 % of the S. aureus strains that were examined exhibited heteroresistance to the mecA gene as well as resistance to oxacillin. The results clearly demonstrated that Ampicillin, Penicillin, amoxicillin, cefazolin, imipenem, and oxacillin are unsuitable choices for the treatment of blood infections in individuals with SCD. “The findings of this study confirmed that all S. aureus isolates were sensitive to gentamicin, and more than 80 % of the isolates were sensitive to ciprofloxacin, moxifloxacin, levofloxacin, clindamycin, and vancomycin.” Further investigation is necessary to explore potential factors that increase the susceptibility of individuals with SCD to bacteremia caused by antibiotic-resistant strains of S. aureus.

Occurrence of Plasmid-Mediated Quinolone Resistance and Carbapenemase-Encoding Genes in Pseudomonas aeruginosa Isolates from Nosocomial Patients in Aguascalientes, Mexico.

Pseudomonas aeruginosa is a leading cause of healthcare-associated infections, which are related to substantial morbidity and mortality. The incidence of Plasmid-Mediated Quinolone Resistance (PMQR) determinants has been previously reported in this bacterium. However, there is limited information regarding the presence of PMQR and carbapenemase-encoding genes simultaneously. This study aims to analyze the prevalence of these determinants on P. aeruginosa strain isolated from clinical patients in the State of Aguascalientes, Mexico. Fifty-two P. aeruginosa isolates from nosocomial patients were collected from Centenario Hospital Miguel Hidalgo. This is a retrospective observational study conducted at a single center. Antibiotic susceptibility was tested using the Vitek-2 system. Only carbapenem-resistant isolates were included in this study. Carbapenemase-encoding genes and PMQR determinants were screened by polymerase chain reaction (PCR). Resistance rates of 100% were found on tigecycline and ceftriaxone. Of the 52 isolates, 34.6% were positive for the qnr genes, 46.2% for the oqxA gene, and 25% for the aac-(6')-lb gene. The most frequent carbapenemase genes found in the samples were blaOXA-51 (42.3%), blaOXA-1 (15.4%), and blaVIM (15.4%). blaOXA-51 co-carrying oqxA was detected in 21.1% of the isolates, blaOXA-51 co-carrying aac-(6')-lb in 11.5%, blaVIM co-carrying aac-(6')-lb in 3.8%, and blaKPC co-carrying oqxA in 5.8%. Systematic surveillance to detect carbapenemase-encoding genes and PMQR determinants, and rational prescription using the last-line drugs could help in preventing the dissemination of multidrug-resistant determinants.

The Microbiological Quality of Raw Ovine Milk in the Banat Region of Romania with a Focus on Escherichia coli and Its Pathogenic Potential and Antimicrobial Resistance.

This study investigated the bacteriological quality of raw ovine milk produced by farms located in the Banat region of Romania. Additionally, the pathogenic potential and antimicrobial resistance of the isolated Escherichia coli strains were evaluated. A total of 95.8% (69/72) of the screened bulk tank milk samples, collected at the farm level immediately after milking, demonstrated appropriate total aerobic mesophilic bacteria (TMB) counts, varying from 3.32 to 6.09 log10 CFU/mL. However, 4.2% (3/72) of the samples were above the regulatory limit of 6.18 log10 CFU/mL. E. coli was identified in 66.6% of the examined samples, and from the total number (n = 48) of isolates, 18.8% harbored the stx2 gene, highlighting pathogenic potential. Antimicrobial susceptibility testing with the Vitek2 system of the isolated E. coli strains revealed resistance against ampicillin (45.8%), gentamicin (20.8%), ticarcillin-clavulanic acid (18.8%), cephalexin (18.8%), amoxicillin-clavulanic acid (8.3%), and trimethoprim-sulfamethoxazole (2.1%). Additionally, 64.6% of the strains showed intermediate resistance against amoxicillin-clavulanic acid, while no resistance was recorded against imipenem. Five (18.5%) strains were multidrug-resistant. This study's results underline hygienic sanitary deficiencies during the milking phase and indicate that raw ovine milk can be contaminated with pathogenic and multidrug-resistant E. coli strains, highlighting a potential risk to public health. Further studies are encouraged to better understand the risk posed to the consumer via the consumption of ovine milk and derived products.

Antibacterial Activity of Silver Nanoparticles Prepared from Camellia Sinensis Extracts in Multi-Drug Resistant Staphylococcus Aureus.

The nano-method has been used as a technique for creating novel, non-traditional antimicrobial agents. This effective method for treating infectious diseases has many advantages over conventional antibiotics, including increased efficacy against species that have developed drug resistance, and the ability to circumvent the development of resistance that disrupts a number of biological pathways. As a result, the objective of this study was to synthesize and characterize silver nanoparticles using phenolic compounds obtained from Camellia sinensis . The nanoparticles were then used as antibacterial agents on the multidrug resistant Staphylococcus aureus, as well as, biofilm formation mechanism were also investigated. Ten isolates of Staphylococcus aureus were acquired from the labs of the University of Baghdad's Genetic Engineering and Biotechnology Institute. The VITEK-2 system was used to confirm the diagnosis. Aqueous and methanolic extracts of Camellia sinensis leaves were used to create silver nanoparticles and obtain CAgNPs, which were then characterized using Atomic Fluorescence Microscopy (AFM), X-ray diffractometer (XRD), and Zeta potential analyzers. The extracts were put through a series of assays, including High-performance liquid chromatography (HPLC), antibacterial activity assessments, and the microtiter plate method to determine the lowest inhibitory concentration (MIC) and antibiofilm formation. Both aqueous and methanolic extracts containing silver nanoparticles included spherical nanoparticles that may be single or combined. The HPLC results showed the presence of two phenolic compounds (gallic acid and caffeine) by comparing their retention durations to those of the reference compounds. The results of the antibacterial activity of (CAgNPs) showed that the methanolic (CAgNPs) extract was more effective than the aqueous (CAgNPs) extract, producing inhibitory zones of 15.67 ± 0.58 and 20.33 ± 0.58 mm, at 375 and 750 ppm respectively, when compared to the aqueous (CAgNPs) extract, which produced inhibitory zones of 12.33 ± 0.58 and 15.67 ± 0.58 mm, respectively. The MIC result also showed that the CAgNPs methanolic extract was more effective than the CAgNPs aqueous extract. The MIC of the CAgNPs methanolic extract on S. aureus isolates were 11.718 and 23.43 µg/ml, while the MIC of the CAgNPs aqueous extract on all S. aureus isolates were 46.87 µg/ml except isolate No. 3 and 6 which was 11.718 and 93.75 µg/ml respectively. Additionally, The anti-biofilm in S. aureus was increased when CAgNPs methanolic extract were used compared with the CAgNPs aqueous extract, the CAgNPs methanolic extract inhibited 80%, 90% and 100% of the biofilm formation of S. aureus in 23.43, 46.87 and 93.75 µg/ml respectively, while the anti-biofilm activity of the CAgNPs aqueous extract on S. aureus isolates was 80% and 100% of the biofilm formation in 46.87 and 93.75 µg/ml respectively. The methanolic and aqueous leaves extracts from Camellia sinensis are a successful method for producing CAgNPs. The synthesized CAgNPs also have significant antibacterial activity against S. aureus, depending on the concentrations, and inhibit S. aureus biofilm formation.

Colistin, Meropenem-Vaborbactam, Imipenem-Relebactam, and Eravacycline Testing in Carbapenem-Resistant Gram-Negative Rods: A Comparative Evaluation of Broth Microdilution, Gradient Test, and VITEK 2.

This study aimed to evaluate and compare the performance of different assays for antimicrobial susceptibility testing (AST) and minimum inhibitory concentration (MIC) determination for reserve antibiotics in carbapenem-resistant Enterobacterales (CREs), Pseudomonas aeruginosa (CRPAs), and Acinetobacter baumannii (CRABs). An analysis was conducted on 100 consecutive isolates: 50 CREs, 35 CRPAs, and 15 CRABs. Sensititre broth microdilution was used as a reference standard to evaluate the performance of VITEK 2 card AST-XN24 (bioMérieux), the respective gradient tests (bioMérieux), and UMIC colistin broth microdilution test strips (Bruker Daltonics). Errors, essential agreement (EA), and categorical agreement of MICs for colistin (COL), meropenem-vaborbactam (MVB), imipenem-relebactam (IRL), and eravacycline (ERV) were assessed. The agreement between both of the COL broth microdilution (BMD) methods was perfect (100/100). The gradient test and VITEK 2 analysis yielded comparable EA rates (92/100 and 72/79, respectively), with the latter not registering any very major errors (VMEs). The MVB gradient test achieved EA in 66 of 85 isolates and VITEK 2 in 70/85. For IRL, EA was reached in 69 and 64 of 85 cases by gradient test and VITEK 2 analysis, respectively. The ERV gradient test yielded false results in nearly all (12/15) CRABs but achieved EA in 46 of 50 CREs. The VITEK system recorded EA for ERV in 60 of 65 isolates. We observed substantial variability in the measured MICs between BMD and the alternative methods. In only a few constellations, VITEK 2 or gradient tests could substitute the reference method. BMD is the method of choice for COL analysis, with VITEK 2 representing an alternative method for CRPA testing. Alternative methods for MVB did not provide reliable results, except for Enterobacterales, when tested with the gradient test. However, resistant results need to be confirmed by BMD. Only BMD can be used for IRL MIC determination. VITEK 2 was mostly accurate in measuring ERV MICs, while the corresponding gradient test yielded reliable results exclusively in CREs. It is essential that laboratories are aware of which testing method provides reliable results for each combination of microorganisms and reserve antibiotics.

Phenotypic, Molecular Identification and Virulence Assessment of Cronobacter spp Isolated from Clinical Samples of Children Under Two Years in Mosul City/Iraq

Cronobacter spp. is known to be a foodborne causative agent for a variety of diseases in both humans and animals. This study focused on isolating Cronobacter species from 150 clinical samples of children under two years (60 from blood and stool samples, 30 from CSF samples) collected from Ibn al Atheer and Al Khanssa Hospitals. The phenotypic identification of bacterial isolates were performed through culture, microscopic, and biochemical examinations and vitek-2 system. The results revealed a mixture of bacteria in 6.5% of the clinical specimens. The identity of isolates was 99% using the vitek-2 system for identifying Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, and Cronobacter pulveris. Out of 150 specimens there were 8 (5.33 %) of specimens gave positive for Cronobacter spp which included: C. sakazaki 2/60 blood specimens (3.3%), C. sakazaki 2/60 stool specimens (3.3%), C. malonaticus 2/60 stool specimens (3.3%), 1/60 C. muytjensii and C. pulveris 1/60 (3.3%). The eight isolates phenotypically identified as Cronobacter were confirmed at the molecular level through 16S rRNA sequencing and submitted to NCBI under the accession numbers (OR825874, OR825875, PP126443, PP126444, PP126445, PP126455). The virulence profile of these isolates showed that 7/8 (87.5%) of Cronobacter strains exhibited haemolysin activity, 5/8 strains (62.5%) were able to produce the protease enzyme and 2/8 (25%) of Cronobacter strains were positive for lipase and lecithinase. All strains lacked the ability to produce a slime layer. The results also showed the ability of Cronobacter strains to produce biofilm by using the tube method and microtiter plate method but with different levels.

Distribution and Antimicrobial Resistance of Complicated Intraabdominal Infection Pathogens in Two Tertiary Hospitals in Egypt.

Background: Management of complicated intraabdominal infections (cIAIs) requires containment of the source and appropriate initial antimicrobial therapy. Identifying the local data is important to guide the empirical selection of antimicrobial therapy. In this study, we aimed to describe the pathogen distribution and antimicrobial resistance of cIAI. Methods: In two major tertiary care hospitals in Egypt, we enrolled patients who met the case definition of cIAI from October 2022 to September 2023. Blood cultures were performed using the BACTAlert system (BioMerieux, Marcy l'Etoile, France). A culture of aspirated fluid, resected material, or debridement of the infection site was performed. Identification of pathogens and antimicrobial susceptibility testing were conducted by the VITEK-2 system (BioMerieux, Marcy l'Etoile, France). Gram-negative resistance genes were identified by PCR and confirmed by whole bacterial genome sequencing using the Nextera XT DNA Library Preparation Kit and sequencing with the MiSeq Reagent Kit 600 v3 (Illumina, USA) on the Illumina MiSeq. Results: We enrolled 423 patients, 275 (65.01%) males. The median age was 61.35 (range 25-72 years). We studied 452 recovered bacterial isolates. Gram-negative bacteria were the vast majority, dominated by E. coli, followed by Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Proteus mirabilis (33.6%, 30.5%, 13.7%, 13%, and 5.4%, respectively). High rates of resistance were detected to third- and fourth-generation cephalosporins and fluoroquinolones. No resistance was detected to colistin. Resistance to amikacin and tigecycline was low among all isolates. Resistance to meropenem and ceftazidime/avibactam was moderate. ESBL genes were common in E. coli and K. pneumoniae. CTX-M15 gene was the most frequent. Among Enterobacterales, blaOXA-48 and blaNDM were the most prevalent carbapenemase genes. Pseudomonas aeruginosa isolates harbored a wide variety of carbapenemase genes (OXA, NDM, VIM, SIM, GIM, SPM, IMP, AIM), dominated by metallo-beta-lactamases. In 20.6% of isolates, we identified two or more resistance genes. Conclusion: High resistance rates were detected to third- and fourth-generation cephalosporins and fluoroquinolones. Amikacin and tigecyclines were the most active antimicrobials. Our data call for urgent implementation of antimicrobial stewardship programs and reinforcement of infection control.

Gram-Negative Bloodstream Infections in a Medical Intensive Care Unit: Epidemiology, Antibiotic Susceptibilities, and Risk Factors for in-Hospital Death.

Gram-negative bloodstream infection (GNBI) poses a serious threat to critically ill patients. This retrospective study aimed to uncover drug resistance of pathogens and the GNBI effect on in-hospital death and distinguish death risk factors in a medical intensive care unit (ICU). A retrospective study of all GNBI patients in the medical ICU of the Third Xiangya Hospital over 9 nine years was conducted. Blood samples were performed by a BACTEC 9240 system, MALDI-TOF MS, Bruker and Vitek-2 system. Logistic regression was used for analyzing risk factors for death. Seventy-five episodes of GNBI developed in 68 (1.4%) out of 4954 patients over a span of 9 years. The most frequently isolated bacterium was Klebsiella pneumoniae, with the lungs as the predominant source of GNBI. The resistance rate of Gram-negative bacteria to polymyxin B was 11.6% after excluding those intrinsically resistant non-fermentativebacteria. All Enterobacter spp. were susceptible to ceftazidime/avibactam. Thirty-three (48.5%) patients underwent inappropriate empirical antibiotic treatment and 48 (70.6%) patients died during the hospitalization. Multivariate logistic regression analysis identified that lymphocyte count at GNBI onset ≤0.5×109/L, invasive mechanical ventilation, and septic shock were related to in-hospital death. Body mass index ≥23 and appropriate empirical antibiotic use after GNBI were negatively associated with in-hospital death. GNBI was a frequent complication among patients in the medical ICU. This study underscored the presence of diverse factors that either heightened or attenuated the risk of in-hospital death.

First Detection of High-Level Aminoglycoside-Resistant Klebsiella pneumoniae and Enterobacter cloacae Isolates Due to 16S rRNA Methyltransferases with and Without blaNDM in Uruguay.

The increase in antimicrobial resistance includes emerging mechanisms such as 16S ribosomal RNA methylases, which confer high-level resistance to aminoglycosides. In this regard, the most predominant genes observed worldwide are rmtB and armA, but their presence in Uruguay is unknown. We describe the genomic characterization of isolates carrying rmtB and rmtC, together with blaNDM-5 and blaNDM-1, respectively, and rmtD in our country. Methology: Five isolates from patients admitted to three hospitals were studied. Identification and antibiotic susceptibility testing were performed using the Vitek2 System. Whole Genome Sequencing was conducted, and hybrid assembly was performed with Unicycler. In silico analysis using the Center for Genomic Epidemiology's tools was undertaken to predict antibiotic resistance determinants, plasmid incompatibility groups, and sequence types. We report three K. pneumoniae ST307 isolates with an IncR plasmid carrying blaNDM-5/blaCTX-M-15/blaTEM-1B/rmtB/dfrA14/dfrA12/sul1/qacEΔ1/ermB/mphA, one K. pneumoniae ST258 harboring an IncC plasmid containing rmtC/blaNDM-1/blaCMY-6/aac(6')-Ib/sul1, and one E. cloacae ST88 isolate with an IncFIB/II plasmid hosting rmtD, within a novel Tn21-like transposon named Tn7825, alongside blaOXA-101/sul1/tet(G)/floR, and a new variant of blaTEM assigned as blaTEM-258. One of the strains, named UH_B2, also carried an IncM1 plasmid encoding qnrE1/blaTEM-1/blaCTX-M-8 associated with ISEcp1. This is the first description of plasmids harboring 16S rRNA methyltransferases in Uruguay. The association and dissemination of diverse antibiotic-resistant genes underpin the health threat they represent, highlighting the lack of available antibiotics effective against multidrug-resistant microorganisms.

Characterization, Antibiotic Susceptibility, and Clonal Analysis of Carbapenem-Resistant Klebsiella pneumoniae From Different Clinical Cases.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is recognized for its great ability to resist prescription drugs and its association with severe infections in humans. This study was designed to evaluate the characteristic resistance spectrum, to characterize the implicated carbapenem-resistant genes (CRGs), and to determine the extent of genetic diversity among Klebsiella pneumoniae isolates from human clinical cases in Duhok province. Methodology: The VITEK-2 system was used to investigate the phenotypic antibiotic susceptibility of 23 K. pneumoniae isolated from distinct human clinical situations, multiplex PCR was used to assign the key common carbapenem-resistant genes (IMP, OXA48-like, bla-NDM, and KPC) in phenotypically carbapenem-resistant isolates, and the Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) assay was utilized to ascertain the clonal associations among those isolates. Phenotypic resistance analysis revealed high resistance rates to various antibiotics, with all isolates exhibiting multidrug resistance (MDR). Coronavirus disease 2019 (COVID-19) patient isolates demonstrated significantly higher resistance compared to other sources. In addition, all isolates showed complete phenotypic resistance to carbapenems, PCR screening for CRGs identified blaOXA-48 as the predominant gene, present in all isolates. Genetic fingerprinting revealed diverse genotypes, with COVID-19 patient isolates exhibiting high similarity, contrasting with maximum diversity in non-COVID-19 clinical isolates.

GENETIC VARIATION OF HOUSEKEEPING GENES IN MULTIDRUG RESISTANT PSEUDOMONAS AERUGINOSA

Background: Pseudomonas aeruginosa is a prominent opportunistic pathogen responsible for nosocomial infections, particularly among immunocompromised individuals. Specific Background: Its ability to develop multiple antibiotic resistance poses a significant clinical challenge, highlighting the need for a deeper understanding of its genetic diversity and virulence factors. Knowledge Gap: While previous studies have explored antibiotic resistance mechanisms, there is limited research on the genetic diversity of Pseudomonas aeruginosa isolates in specific geographic regions, such as Kirkuk. Aims: This study aimed to investigate the genetic diversity of Pseudomonas aeruginosa isolates from clinical samples obtained from Kirkuk Civil Hospitals, utilizing Multi-Locus Sequence Typing (MLST) for genetic analysis. Results: Fourteen P. aeruginosa isolates were confirmed through biochemical tests and the VITEK-2 system, with an alarming 85.71% (12/14) exhibiting antibiotic resistance. Molecular analysis revealed the presence of several housekeeping genes, although some genes did not amplify. Notably, two new serotypes (PS3:id:9797 and PS4:id:9796) were identified and added to the MLST database, along with three new genes registered in NCBI. Phylogenetic analysis indicated a divergent cluster among three isolates. Novelty: This research contributes new insights into the genetic diversity of Pseudomonas aeruginosa, identifying novel serotypes and genes, which are critical for understanding its epidemiology and resistance mechanisms. Implications: The findings underscore the importance of ongoing surveillance of Pseudomonas aeruginosa in clinical settings to inform treatment strategies and public health policies aimed at managing antibiotic resistance and improving patient outcomes.

The Impact of Heavy Metal Ions on Arginase Produced from Pseudomonas aeruginosa Isolated from Sewage

Background: Researchers remain captivated by the impact of heavy metal ions, commonly found in sewage, on arginase synthesis. Objective: The study examines the presence and characteristics of Pseudomonas aeruginosa in 24 sewage samples, identifying 18 isolates (75%). It assesses DNA concentration and purity, utilizes PCR-based methods for subtype analysis and investigates the inhibitory effects of heavy metals on purified enzymes. Methods: Between November 2022 and April 2023, 52 samples were collected from various sewage sites in Baghdad, Iraq. These samples were cultured in brain heart infusion broth and on nutrient agar plates. The isolates were then subjected to laboratory procedures, including biochemical tests, analysis with the Vitek2 system and PCR to identify Pseudomonas aeruginosa. Biochemical testing specifically focused on Pseudomonas aeruginosa. DNA extraction was performed on P.aeruginosa isolates from sewage, which had already been identified through morphological and biochemical tests as P.aeruginosa. This was accomplished by screening for the presence of the 16SrRNA gene using PCR. Result: Metal ion effects on L-arginase were tested at various concentrations (100mM, 50mM, 25mM, 12.5 mM). Metal ions were obtained via the cutting method and added 15 minutes before the substrate. Enzyme activity was measured and the activity ratio (enzyme activity with and without metal ions) calculated. Activity was assessed in the absence of 100% metal ions.Arginase isolated and purified from Pseudomonas aeruginosa from sewage, heavy metals (mercury, cadmium, cobalt) has effect on arginase, mercuric chloride and cadmium chloride inhibit and decrease enzyme activity, while Cobalt chloride increases enzyme activity.