Rapid detection of carbapenemase-producing gram-negative bacteria directly from blood culture bottles for a reliable guided empiric therapy.

Bloodstream infections (BSIs) caused by multidrug-resistant bacteria are a critical life-threatening challenge which necessitates the urgency to trigger life-saving treatment in a timely manner. This study aimed to evaluate the time required for rapid detection of carbapenemase-producing Enterobacterales (CPE) directly from blood culture bottles to optimize empirical treatment of BSI, especially in pediatric and infant patients, using a cost-effective method. This study included 419 Gram-negative bacteria, of which Klebsiella pneumoniae and Escherichia coli were the most common CPE causing BSI in pediatric and neonatal patients. Phenotypic and genotypic resistance of the selected isolates (45 K. pneumoniae and 9 E. coli) were determined by VITEK-2 Compact system and PCR, respectively. BACT/ALERT bottles were spiked with isolates. Finally, colorimetric RESIST-BC assay and Vitek-2 compact system were evaluated for the rapid detection of carbapenem-resistant bacteria directly from positive blood culture bottles. All selected isolates were phenotypically resistant to carbapenems. PCR showed that blaNDM and blaOXA-48 were present in all isolates, blaVIM was present in 44.4%, while blaKPC and blaIMP were entirely absent. The RESIST-BC kit showed good agreement with PCR for blaNDM and blaOXA-48, demonstrating high sensitivity and specificity, but not with blaVIM. These findings point out that RESIST-BC assay demonstrated an exceptionally short detection time for CPE, completing all cases within the first hour after the blood culture bottles flagged positive. It is also superior in providing a clue for clinicians on antibiotic combinations that can be administered, depending on the type of ß-lactamases detected, promptly and efficiently, with low expenses.

The Effect of Citric and Ascorbic Acids as Anti-Biofilm and Anti-Capsular Agents on Multidrug-Resistant Acinetobacter baumannii.

OBJECTIVE: Acinetobacter baumannii (A. baumannii) is an opportunistic bacterium with multiple virulence factors, including capsule and biofilm, and is known for its high drug resistance. Anti-virulence natural substances have been suggested as novel alternatives to conventional antibiotics. We aimed to evaluate the effect of citric and ascorbic acids as anti-biofilm and anti-capsular agents against multidrug-resistant (MDR) A. baumannii clinical isolates. MATERIALS AND METHODS: Twenty-eight A. baumannii MDR isolates were collected from different clinical sources. The minimum inhibitory concentration (MIC) of each agent was estimated. Biofilm formation and capsule were investigated phenotypically in the absence and presence of both agents at ½ and » MICs. The presence of 14 adhesive and nonadhesive virulence genes was investigated. RESULTS: Phenotypically, all the isolates were biofilm producers and were capsulated. The MIC of citric acid ranged from 1.25 to 2.5 mg/mL, while that of ascorbic acid was 3 mg/mL for all isolates. Both agents showed significant reduction in biofilm and capsular thinning. Ascorbic acid showed a dose-dependent effect in both biofilm reduction and capsule thinning unlike citric acid. Four genes, papG23, sfa1, fyuA, and cvaC, were absent among all isolates, while iutA was present in 100% of isolates. Other genes showed different distributions among the isolates. These virulence genes were not correlated to the anti-biofilm effect of both agents. Ascorbic acid was observed to have a better effect than citric acid. This can provide a clue for a better treatment regimen including ascorbic acid against MDR A. baumannii infections.

Bacterial vaginosis, vulvovaginal candidiasis, trichomonal vaginitis and aerobic vaginitis in women from Egypt.

Introduction: Infectious vaginitis is prevalent in developing countries. Most of the females suffer from vaginal infections at least once per lifetime. Due to limited resources, many infections are misdiagnosed or undiagnosed. Good diagnosis of these infections is critically important and will definitely help to guide treatment and prevent recurrence. Methods: A total of 1080 vaginal swabs were collected from symptomatic females. Nugent's score and Amsel's criteria were applied to diagnose bacterial vaginosis (BV). A rapid test was used to identify Gardnerella vaginalis. Trichomonal vaginitis (TV) was diagnosed through microscopic examination. Vulvovaginal candidiasis (VVC) was also identified microscopically and using conventional culture. Finally, aerobic vaginitis (AV) was detected using Donder's scale combined with conventional culture and biochemical tests. Results: There was no statistically significant association between age and type of vaginal infection (p=0.130). Vulvovaginal inflammation, itching and redness were significantly associated with VVC (p≤0.012). BV was detected as single infection in 43.8%, followed by VVC 24.2%. On the contrary, AV and TV were scarcely detected among the participants; 4.9% and 0.5% respectively. Mixed infections between BV and VVC were noted in 26.6%. Conclusions: BV showed the highest prevalence followed by VVC. Mixed infections between BV and VVC were evidently noted, therefore good reliable diagnosis using cost-effective methods is crucial for proper treatment. Aerobic vaginitis showed low prevalence and most of the Streptococcus spp. were isolated from pregnant females. The low prevalence of Trichomonas vaginalis may be due to the dependance on conventional methods for diagnosis, and thus more advanced diagnostic tools are required.

SUMO Modification of Hepatitis B Virus Core Mediates Nuclear Entry, Promyelocytic Leukemia Nuclear Body Association, and Efficient Formation of Covalently Closed Circular DNA.

Persistence of hepatitis B virus (HBV) infection is due to a nuclear covalently closed circular DNA (cccDNA), generated from the virion-borne relaxed circular DNA (rcDNA) genome in a process likely involving numerous cell factors from the host DNA damage response (DDR). The HBV core protein mediates rcDNA transport to the nucleus and likely affects stability and transcriptional activity of cccDNA. Our study aimed at investigating the role of HBV core protein and its posttranslational modification (PTM) with SUMO (small ubiquitin-like modifiers) during the establishment of cccDNA. HBV core protein SUMO PTM was analyzed in His-SUMO-overexpressing cell lines. The impact of HBV core SUMOylation on association with cellular interaction partners and on the HBV life cycle was determined using SUMOylation-deficient mutants of the HBV core protein. Here, we show that the HBV core protein is posttranslationally modified by the addition of SUMO and that this modification impacts nuclear import of rcDNA. By using SUMOylation-deficient HBV core mutants, we show that SUMO modification is a prerequisite for the association with specific promyelocytic leukemia nuclear bodies (PML-NBs) and regulates the conversion of rcDNA to cccDNA. By in vitro SUMOylation of HBV core, we obtained evidence that SUMOylation triggers nucleocapsid disassembly, providing novel insights into the nuclear import process of rcDNA. HBV core protein SUMOylation and subsequent association with PML bodies in the nucleus constitute a key step in the conversion of HBV rcDNA to cccDNA and therefore a promising target for inhibiting formation of the HBV persistence reservoir. IMPORTANCE HBV cccDNA is formed from the incomplete rcDNA involving several host DDR proteins. The exact process and the site of cccDNA formation are poorly understood. Here, we show that HBV core protein SUMO modification is a novel PTM regulating the function of HBV core. A minor specific fraction of the HBV core protein resides with PML-NBs in the nuclear matrix. SUMO modification of HBV core protein mediates its recruitment to specific PML-NBs within the host cell. Within HBV nucleocapsids, SUMOylation of HBV core induces HBV capsid disassembly and is a prerequisite for nuclear entry of HBV core. SUMO HBV core protein association with PML-NBs is crucial for efficient conversion of rcDNA to cccDNA and for the establishment of the viral persistence reservoir. HBV core protein SUMO modification and the subsequent association with PML-NBs might constitute a potential novel target in the development of drugs targeting the cccDNA.

Ceftazidime-Avibactam plus aztreonam synergistic combination tested against carbapenem-resistant Enterobacterales characterized phenotypically and genotypically: a glimmer of hope.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) show rapid global dissemination and pose a significant therapeutic challenge. This study aimed to characterize carbapenemase-producing Klebsiella spp. and Escherichia coli (E. coli) phenotypically and genotypically and evaluate the effect of ceftazidime/ avibactam plus aztreonam combination. METHODS: A total of 219 Klebsiella species and 390 E. coli strains were isolated from clinical samples, in which 80 Klebsiella spp. and 20 E coli isolates were resistant to tested carbapenems (imipenem, ertapenem, meropenem) by disk diffusion/broth dilution method and Vitek-2 compact system. MASTDISCS Combi Carba plus discs and real time PCR were used to determine type of carbapenemase phenotypically and genotypically, respectively. Interestingly, the synergistic effect between ceftazidime-avibactam (E-test) and aztreonam (disc) was tested against the CPE isolates. RESULTS: Out of the carbapenem-resistant isolates, 76.25% Klebsiella spp. isolates were extensively drug-resistant (XDR) while 18.75% were pan drug-resistant (PDR), and 5% were multidrug-resistant (MDR). Regarding E. coli, 5% were PDR, 20% were MDR and 75% were XDR. More than one carbapenemase gene was detected in 99% of the isolates. In comparison between MAST-Carba plus discs and PCR results, sensitivity and specificity were (85.42-97.92%) in Klebsiella spp., and (69.64-100%) in E. coli, respectively. Moreover, a strong association was detected between both test results among Klebsiella spp. (p < 0.001) and E. coli (p = 0.012) isolates. Finally, ceftazidime-avibactam and aztreonam combination showed a synergistic effect in 98.8% of Klebsiella spp. and 95% of E coli. All 16 PDR isolates showed synergy. CONCLUSION: This synergistic effect spots the light on new therapeutics for XDR and PDR CPE.

Characterisation of Methicillin-Resistant Staphylococcus aureus from Alexandria, Egypt.

The present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt's second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+fus] (n = 6), CC1-MRSA-[V+fus+tir+ccrA/B-1] (PVL+) (n = 5) as well as CC1-MRSA-[V+fus+tir+ccrA/B-1] and CC1153-MRSA-[V+fus] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCCmec V or VI elements that included the fusidic acid resistance gene fusC. The SCCmec [V+fus+tir+ccrA/B-1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ, aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCCmec elements comprising mecA and fusC. This is an unsettling trend, but more MRSA typing data from Egypt are required.

Effect of Ruta graveolens Extract on the Major Virulence Factors in Methicillin Resistant Staphylococcus aureus.

Purpose: Rising Antibiotic Resistance has put the world in real threat. Methicillin resistant Staphylococcus aureus (MRSA), is a predominant cause of suppurative chronic skin and soft-tissue infections. Novel insights have focused the light on plant extracts. In this study, Ruta graveolens ethanolic active extract was tested for its potential anti-virulence activities in MRSA. Materials and Methods: A total of 100 MRSA strains causing skin and soft tissue infections were isolated and antibiotic susceptibility testing was done. Ability to form biofilm was tested phenotypically. Furthermore, the antimicrobial activity of Ruta graveolens was evaluated followed by detection of its Minimum inhibitory concentration (MIC). The inhibitory activity of this extract on biofilm formation was investigated. Afterwards, we investigated its effect on the transcription of biofilm-related genes and mecA gene. Results: All tested isolates were sensitive to Vancomycin and Linezolid while high resistance was noted with both Fusidic acid (83%) and Gentamicin (68%). (83%) of the isolates were biofilm producers. Ruta graveolens extract showed strong antimicrobial activity against the MRSA strains with MIC 0.78 mg/mL. At subinhibitory concentration (1/2 MIC), the extract had high biofilm inhibitory effects with mean inhibition (70%). Moreover, transcriptional analysis results showed that the mean percentages of inhibition in expression of mecA, icaA and icaD genes were 52.3%, 34.8% and 33.7%, respectively, in which all showed statistically significant difference (p ≤ 0.05). Conclusion: The current study proposes the ability of Ruta graveolens extract to reduce the biofilm formation and antibiotic resistance of MRSA through downregulation of some biofilm forming genes and mecA gene which confers resistance to B-lactam antibiotics. This may decrease our reliance on antibiotics and improve our ability to effectively treat biofilm-related skin and soft-tissue infections caused by MRSA.