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.

Human coronavirus OC43 and other respiratory viruses from acute respiratory infections of Egyptian children.

Respiratory infections have a significant impact on health worldwide. Viruses are major causes of acute respiratory infections among children. Limited information regarding its prevalence in Egypt is available. This study investigated prevalence of 10 respiratory viruses; Adenovirus, influenza A, B, respiratory syncytial virus (RSV), Parainfluenza virus (PIV)type 1-4, enterovirus, and human coronavirus OC43 (HCoV-OC43) among children in Alexandria, Egypt presenting with acute lower respiratory tract infections.The study was conducted on children <14 years of age selected from ElShatby Pediatric Hospital, Alexandria University, Egypt. One hundred children presenting during winter season with influenza-like illness were eligible for the study. Oropharyngeal swabs were collected and subjected to viral RNA and DNA extraction followed by polymerase chain reaction.Viral infections were detected in 44% of cases. Adenovirus was the most common, it was found in 19% of the patients. Prevalence of PIV (3 and 4) and enterovirus was 7% each. Prevalence of RSV and HCoV-OC43 was 5% and 3% respectively. Two percentage were Influenza A positive and 1% positive for influenza B. Mixed viral infection was observed in 7%.To the best of our knowledge, this is the first report of the isolation of HCoV-OC43 from respiratory infections in Alexandria, Egypt.

Emergence of blaVEB and blaGES among VIM-producing Pseudomonas aeruginosa clinical isolates in Alexandria, Egypt.

Thirty-three Pseudomonas aeruginosa isolates, resistant to one or more ß-lactams, were included in this study. Identification of tested strains was confirmed using MALDI-TOF/MS. Phenotypic and genotypic ß-lactamase patterns were investigated. Most of the isolates were resistant to carbapenems (32 out of 33) and to the extended-spectrum cephalosporins (ESC) (30 out of 33). Phenotypically, the production of extended-spectrum beta-lactamase (ESBL), metallo-ß-lactamases (MBL), and carbapenemases was detected in 10, 23, and 9 isolates, respectively. However, AmpC hyperproduction was not phenotypically detected among all isolates. Genotypically, ESBL and MBL encoding genes were detected in 23 and 27 isolates, respectively. Altogether 27 strains were detected as blaVIM positive and 16 strains carried blaOXA-10 gene. To the best of our knowledge, this is the first report of P. aeruginosa clinical isolates harboring blaVEB together with blaGES in Egypt, where 5 of our 30 ESC-resistant isolates showed this genotype. Our results confirmed that resistance of P. aeruginosa isolates to ß-lactam antibiotics is mediated via multiple ß-lactamases belonging to different molecular classes. To the best of our knowledge, this is the first report of blaVEB among P. aeruginosa clinical isolates from Egypt. Ten isolates harbored blaVEB and five of them co-harbored blaVEB together with blaGES, blaVIM, and blaOXA-10.

Detection of low-intensity Schistosoma mansoni infection by Percoll sedimentation and real-time PCR techniques in a low-endemicity Egyptian setting.

OBJECTIVE: To evaluate the performance of Percoll sedimentation and real-time polymerase chain reaction (PCR) for the detection of S. mansoni cases previously tested as negative by Kato-Katz technique in two low-endemic areas in Alexandria, Egypt, Abis 4 and 8 villages. METHODS: Stool samples of 824 primary schoolchildren were examined by Kato-Katz technique (three slides of 41.7 mg each). After obtaining the results of this survey, stool samples were recollected from a subset of 150 students, who gave negative results after Kato-Katz. These samples were microscopically examined after the concentration with Percoll technique. Part of the 150 negative stool samples and five positive samples (used as controls) were kept at -20 °C and further processed by SYBR Green PCR. RESULTS: Prevalence of S. mansoni infection as determined by three Kato-Katz thick smears was 1.82% (15 cases). Three more cases tested positive by Percoll sedimentation among the 150 samples that were negative by Kato-Katz. Specific amplification by SYBR Green PCR was noted in all positive controls and in three cases of Kato-Katz-negative samples, two of which were also positive by Percoll. CONCLUSION: Percoll sedimentation and SYBR Green PCR proved useful in detecting low-intensity S. mansoni infections in low-endemicity areas in Egypt.

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.

Hepatitis C virus core protein triggers hepatic angiogenesis by a mechanism including multiple pathways.

Chronic hepatitis C virus (HCV) infection is associated with the production of serum cytokines, including transforming growth factor (TGF)-beta2. Despite the occurrence of hepatic angiogenesis in liver conditions, the role of HCV proteins in this context is currently unknown. We demonstrated that the development of hepatic neoangiogenesis in patients infected with HCV is associated with the expression of TGF-beta2 and vascular endothelial growth factor (VEGF) and with activation of endothelial cells, as evidenced by CD34 expression. The analysis of liver biopsies of HCV-positive and HCV-negative patients using immunostaining showed significant elevation of TGF-beta2, VEGF, and CD34 expression in patients who were HCV-positive. Using an HCV established culture system, we confirmed further the production of both TGF-beta2 and VEGF proteins, in the hepatoma cell lines HepG2 and Huh7 by transfection with full-length HCV RNA (JFH1) or by the regulated expression of core. In addition, regulated expression of core protein in HepG2 or Huh7 cells was found to induce expression and activation of the transcription factor E2F1 and apoptosis signal-regulating kinase 1 (ASK1), activation of c-jun-N-terminal kinase (JNK) and p38, and extracellular-regulated kinase (ERK), and transcription factors activator protein 1 (AP-1), activating transcription factor 2 (ATF-2), cyclic adenosine monophosphate response element binding (CREB), E2F1, hypoxia inducing factor 1 alpha (HIF-1alpha), and specificity protein 1. Furthermore, data obtained from inhibitor experiments revealed the importance of E2F1 and ASK1 in the modulation of core-induced activation of JNK and p38 pathways and suggested an essential role for JNK, p38, and ERK pathways in the regulation of core-induced production of TGF-beta2 and VEGF proteins. Thus, our data provide insight into the molecular mechanisms whereby core protein mediates the development of hepatic angiogenesis in patients with chronic HCV infection.

Potential Co-prevalence of Plasmid-Mediated Quinolone Resistance Determinant qepA and 16S rRNA Methylase rmtB among E.coli Clinical Isolates from Alexandria- Egypt.

BACKGROUND: Fluoroquinolone-resistant Gram-negative pathogens have been increasingly reported from most regions of the world over the last decade. A new plasmid-mediated fluoroquinolone efflux pump gene (qepA) is known to be associated with the 16S rRNA methylase gene (rmtB) that confers resistance to aminoglycosides. AIM: To investigate the potential co-prevalence of qepA and rmtB genes in Escherichia coli (E. coli) clinical isolates collected from Egyptian medical facilities. MATERIAL AND METHODS: A total of 225 non-duplicate E. coli isolates were collected between 2008 and 2009 from two hospitals in Alexandria. Isolates were initially tested for their antibiotic susceptibility by disc diffusion method. Isolates exhibited quinolone and aminoglycosides co-resistance profile were screened for the presence of qepA and rmtB genes. The effect of efflux pump inhibitor, phenylarginine-beta-naphthylamide (PAßN) on the minimum inhibitory concentration (MIC) of ciprofloxacin, levofloxacin and gentamicin against these strains was tested and log activity index was calculated. Using checkerboard titration method, the combinations of gentamicin with ciprofloxacin against the strains harboring qepA and rmtB genes were tested and the fractional inhibitory concentrations (FIC) were calculated. RESULTS: Forty-five E. coli isolates exhibited quinolone and aminoglycosides co-resistance profile. Of them, two E. coli isolates were positive for qepA, and three harbored rmtB genes. No association between both genes was detected. The calculated log activity index revealed a reduction in MIC of the fluoroquinolones with PAßN but not of gentamicin. FIC calculated here for gentamicin/ciprofloxacin combinations reflected either antagonism or indifference against the strains harboring qepA and rmtB genes. CONCLUSION AND RECOMMENDATIONS: qepA as well as rmtB genes-carrying E. coli strains could become a greater nosocomial infection problem with appropriate foci of selective pressure. Therefore, public health support for active surveillance for plasmid mediated fluoroquinolones, aminoglycosides resistance determinants among clinical E. coli isolates should be encouraged. In addition, the effect of efflux pumps needs to be considered in the design of future antibiotics as their synergistic role may pave the way to novel combination therapies that could be used against these strains.

Fecal carriage of ESBL-producing Escherichia coli in Egyptian patients admitted to the Medical Research Institute hospital, Alexandria University.

Commensal ESBL-producing E. coli represent a reservoir for resistance genes therefore, their detection is crucial to restrain the spread of beta-lactam resistance. Hence, the aim of the present study was phenotypic and genotypic characterization of commensal ESBL-producing E. coli obtained from the stool of patients at the time of admission and at the time of discharge from the Medical Research Institute hospital. A total of 70 E. coli isolates were collected from 35 patients and were categorized into Group A (samples obtained on admission) and Group B (samples obtained at the time of discharge). Phenotypically, 30 isolates were ESBL producers (40% of E. coli isolates collected on admission and 45.7% of the strains obtained at the time of discharge were ESBL producers). Most of them harbored one to three plasmids with sizes ranging from one kbp to ten kbp. Upon genotypic investigation, bla CTX-M was the most detected gene in 80% of ESBL strains, followed by bla TEM in 53.3% and the least detected was bla SHV in only 13.3%. By comparing group A and group B, ten patients were found to carry commensal ESBL-producing E. coli, in two patients these isolates carried ESBL genes that were identical on admission and on discharge. However, in eight patients, these isolates carried different ESBL genes, which were newly harbored during hospital stay. The high abundance of MDR commensal E. coli 48.57% together with the presence of 42.86% ESBL-producing commensal E. coli among our isolates represents an alarming threat, as they are frequently associated with the increased risk of infection, higher costs and longer hospital stay.

Assessing the marginal error in diagnosis and cure of Schistosoma mansoni in areas of low endemicity using Percoll and PCR techniques.

OBJECTIVE: The Kato-Katz technique misses a proportion of low-intensity schistosomiasis cases. We set out to estimate the rate of false negative results after Kato-Katz. METHOD: Comparison with the more sensitive Percoll technique and polymerase chain reaction (PCR). RESULTS: Amongst Kato-Katz negative Schistosoma mansoni specimens in a hypoendemic area, Percoll detected 11% positive cases and PCR detected 23%. After treatment, the cure rate was 97% by Kato-Katz, 90% by Percoll and 71% by PCR. Results of Kato-Katz and Percoll showed moderate agreement, all cases positive by Kato-Katz were positive by Percoll. However, PCR showed some negative results amongst cases considered sure positives. CONCLUSION: The Percoll technique seems to be the technique of choice for diagnosis of S. mansoni in low intensity areas and after treatment.

Induction of high-molecular-weight (HMW) tumor necrosis factor(TNF) alpha by hepatitis C virus (HCV) non-structural protein 3 (NS3) in liver cells is AP-1 and NF-kappaB-dependent activation.

Chronic infection of hepatitis C virus (HCV)-infected patients is associated with the production of serum and interhepatic inflammatory cytokines including tumor necrosis factor alpha (TNF-alpha). In this study, we delineated part of the mechanism whereby HCV induces the synthesis of TNF-alpha in human liver cell lines HepG2 and Huh7. HepG2 transiently transfected with the full-length HCV cDNA expressed high-molecular-weight (HMW) TNF-alpha mRNAs, which were absent in the control cells. In addition tightly regulated expression of HCV NS3 in both HepG2 and Huh7 was found to induce the expression of HMW mRNAs and subsequently the production of biologically active TNF-alpha. Interestingly, the expression of NS3 protein in HepG2-NS3 or in Huh7-NS3 resulted in the activation of kinase (IKK-alpha) of NF-alphaB inhibitor (IalphaB) and in the enhancement of the DNA-binding activity of the nuclear transcription factor NF-kappaB. The inhibition of the transcription of TNF-alpha mRNAs and subsequently TNF-alpha production following the treatment of HepG2-NS3 or Huh7-NS3 transfectants with the inhibitor of NF-kappaB, Bay 11-7082, suggesting the importance of NF-kappaB for the regulation of NS3-mediated TNF-alpha expression in HepG2 and HeLa cells. Interestingly, data obtained from luciferase assays, in liver and in non-liver cells showed the contribution of NS3 protein in the regulation of TNF-alpha promoter through the activation of AP-1 and NF-kappaB. Our data indicate that the intrahepatic TNF-alpha production induced by HCV is transcriptionally up-regulated by HCV NS3. Therefore, HCV NS3 may have a potential role in the induction of intrahepatic inflammatory processes that occur during acute and chronic hepatitis C.

Activation of RB/E2F signaling pathway is required for the modulation of hepatitis C virus core protein-induced cell growth in liver and non-liver cells.

Hepatitis C virus (HCV) core protein is a multifunctional protein that affects transcription and cell growth in vitro and in vivo. Here, we confirm the proliferative activities of core protein in liver and non-liver cells and delineate part of the mechanism whereby core protein promotes cell growth. We show that core protein suppresses the expression of tumor suppressor protein p53 and cyclin-dependent kinase (CDK) inhibitor p21 and enhances the activation of cyclin-dependent kinase 2 (CDK2), the phosphorylation of retinoblastoma (Rb), the activation of the transcription factor E2F-1, and the expression of E2F-1 and S phase kinase-interacting protein 2 (SKP2) genes. Pretreatment of core protein-expressing cells with the inhibitor of CDK2, Butyrolactone I, abolished the phosphorylation of Rb, the activation of E2F-1, and inhibited the expression of E2F-1 gene and cell growth induced. Consistent with these findings, we define a new signaling pathway whereby the HCV core protein mediates cell growth in infected cells.

Community-acquired methicillin-resistant Staphylococcus aureus from skin and soft tissue infections (in a sample of Egyptian population): analysis of mec gene and staphylococcal cassette chromosome.

BACKGROUND: Staphylococcus aureus has been recognized as an important pathogen associated with inpatients and community infections. Community-acquired methicillin-resistant S. aureus (CA-MRSA) infections commonly present as skin and soft-tissue infections (SSTIs). Treatment often includes incision and drainage with or without adjunctive antibiotics. OBJECTIVES: This study aimed to identify CA-MRSA infections both phenotypically and genotypically, to determine their spectrum of antibiotic resistance, and to establish the best scheme for molecular distinction between hospital-acquired MRSA (HA-MRSA) and CA-MRSA by staphylococcal cassette chromosome mec (SCCmec) typing and detection of Panton Valentine leukocidin (PVL). MATERIALS: 50 swabs, from skin and soft tissue of infected lesions of outpatients attending the dermatology department of the Medical School, Alexandria University, were collected. Additionally, a nasal swab was taken from every participant. METHODS: Collection of swabs from the infected skin and soft tissues, followed by laboratory testing to phenotypically and genotypically identify MRSA. Also, nasal swabs were taken from every patient to identify MRSA colonization. RESULTS: Staphylococcus aureus strains were identified in 38 (76%) of the 50 clinical isolates. 18 (47.37%) out of the 38 S. aureus strains were resistant to oxacillin and cefoxitin discs, were penicillin binding protein 2a (PBP2a) producers, and were initially diagnosed as MRSA. All of the 18 strains were definitively diagnosed as MRSA by mecA gene detection using real time PCR, while only six (33.33%) strains were PVL positive. Using the sets of primers of Zhang et al.: nine (50%) out of the 18 CA-MRSA strains were SCCmec type V, and one (5.56%) was SCCmec type IVc. Then, using the set of primers by Oliveira et al., two (25%) out of the eight untypable MRSA strains were found to be SCCmec type IV, and six (75%) remained untypable. CONCLUSIONS: CA-MRSA must be considered when treating skin and soft tissue infections, especially in developing countries. Empirical use of agents active against CA-MRSA is warranted for patients presenting with serious SSTIs.

Community-acquired methicillin-resistant Staphylococcus aureus from skin and soft tissue infections (in a sample of Egyptian population): analysis of mec gene and staphylococcal cassette chromosome

BACKGROUND: Staphylococcus aureus has been recognized as an important pathogen associated with inpatients and community infections. Community-acquired methicillin-resistant S. aureus (CA-MRSA) infections commonly present as skin and soft-tissue infections (SSTIs). Treatment often includes incision and drainage with or without adjunctive antibiotics. OBJECTIVES: This study aimed to identify CA-MRSA infections both phenotypically and genotypically, to determine their spectrum of antibiotic resistance, and to establish the best scheme for molecular distinction between hospital-acquired MRSA (HA-MRSA) and CA-MRSA by staphylococcal cassette chromosome mec (SCCmec) typing and detection of Panton Valentine leukocidin (PVL). MATERIALS: 50 swabs, from skin and soft tissue of infected lesions of outpatients attending the dermatology department of the Medical School, Alexandria University, were collected. Additionally, a nasal swab was taken from every participant. METHODS: Collection of swabs from the infected skin and soft tissues, followed by laboratory testing to phenotypically and genotypically identify MRSA. Also, nasal swabs were taken from every patient to identify MRSA colonization. RESULTS: Staphylococcus aureus strains were identified in 38 (76%) of the 50 clinical isolates. 18 (47.37%) out of the 38 S. aureus strains were resistant to oxacillin and cefoxitin discs, were penicillin binding protein 2a (PBP2a) producers, and were initially diagnosed as MRSA. All of the 18 strains were definitively diagnosed as MRSA by mecA gene detection using real time PCR, while only six (33.33%) strains were PVL positive. Using the sets of primers of Zhang et al.: nine (50%) out of the 18 CA-MRSA strains were SCCmec type V, and one (5.56%) was SCCmec type IVc. Then, using the set of primers by Oliveira et al., two (25%) out of the eight untypable MRSA strains were found to be SCCmec type IV, and six (75%) remained untypable. CONCLUSIONS: CA-MRSA must be considered when treating skin and soft tissue infections, especially in developing countries. Empirical use of agents active against CA-MRSA is warranted for patients presenting with serious SSTIs.

Activation of c-Jun NH2-terminal kinase (JNK) signaling pathway is essential for the stimulation of hepatitis C virus (HCV) non-structural protein 3 (NS3)-mediated cell growth.

Hepatitis C virus (HCV) non-structural protein 3 (NS3) has been shown to affect cellular functions and is thought to contribute to the development of HCV-related hepatocarcinogenesis. In this study, we delineated part of the mechanisms whereby NS3 protein stimulates cell growth in liver (HepG2) and non-liver (HeLa) cells. The expression of NS3 protein enhanced cell growth, c-jun NH(2)-terminal kinase (JNK) activation, DNA binding activities of the transcription factors AP-1 and ATF-2, and c-jun expression, but not the activation of extracellular signal-regulated kinase (ERK) or p38(MAPK). Whereas co-expression of NS3 with its cofactor NS4A inhibited NS3-mediated cell growth without to influence NS3-mediated JNK activation, or to affect the basal activities of ERK or p38(MAPK). Pre-treatment of NS3 protein-expressing cells with JNK inhibitor, SP600125, abolished activation of AP-1 and ATF-2 and inhibited c-jun expression and induced cell growth, suggesting that JNK activation is essential for the stimulation of NS3-mediated cell growth.