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Sawsan Mohammed Kareem,1 Israa MS Al-kadmy,1,2 Saba S Kazaal,1 Alaa N Mohammed Ali,1 Sarah Naji Aziz,1 Rabab R Makharita,3 Abdelazeem M Algammal,4 Salim Al-Rejaie,5 Tapan Behl,6 Gaber El-Saber Batiha,7 Mohamed A El-Mokhtar,8 Helal F Hetta8,9 1Branch of Biotechnology, Department of Biology, College of Science, University of Mustansiriyah, Baghdad, Iraq; 2Faculty of Science & Engineering, School of Engineering, University of Plymouth, Plymouth, PL4 8AA, UK; 3Botany Department, Faculty of Science, Suez Canal University, Ismailia, 41522, Egypt; 4Department of Bacteriology, Immunology and Mycology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt; 5Department of Pharmacology & Toxicology, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; 6Chitkara College of Pharmacy, Chitkara University, Rajpura, Punjab, India; 7Department of Pharmacology and Therapeutics, Faculty of Veterinary Medicines, Damanhour University, Damanhour, 22511, Egypt; 8Department of Medical Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, 71515, Egypt; 9Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, USACorrespondence: Helal F HettaUniversity of Cincinnati College of Medicine, Department of Internal Medicine, PO Box 670595, Cincinnati, OH, 45267-0595, USAEmail helal.hetta@uc.eduIsraa MS Al-kadmyFaculty of Science & Engineering, School of Engineering, University of Plymouth, Drake Circus, Plymouth, PL4 8AA, United KingdomEmail israa.al-kadmy@plymouth.ac.ukBackground and Aim: Recently, the extensive use of quinolones led to increased resistance to these antimicrobial agents, with different rates according to the organism and the geographical region. The aim of this study was to detect the resistance rate of Klebsiella pneumoniae Iraqi isolates toward quinolone antimicrobial agents, to determine genetic mutations in gyrA and parC, to screen for efflux-pump activity, and to screen the presence of plasmid-mediated quinolone resistance (PMQR) genes.Methods: Forty-three K. pneumoniae isolates were confirmed phenotypically and genotypically by Vitek 2 system and species specific primers by PCR using the targeting rpo gene followed by sequencing. Antibiotic susceptibility test was carried out using disc diffusion method. Quinolone resistant isolates were subjected to ciprofloxacin MIC testing, and cartwheel method to screen for efflux pump activity. The presence of the plasmid mediated quinolone resistance genes qepA, qnrB, qnrS, and aac(6)Ib was tested by PCR. Sequencing of gyrA and parC was performed.Results: We observed a high rate of resistance to ceftriaxone, gentamicin ciprofloxacin, and levofloxacin. Low rate of resistance was detected against amikacin and azithromycin. Ciprofloxacin MIC results revealed that 96.1% of the isolates had MICs > 256 μg/mL, 83.4% had MICs > 512 μg/mL while 34.6% had MIC > 1024 μg/mL. Testing of isolates against ciprofloxacin mixed with EtBr at various concentrations resulted in decreased resistant. Sequencing results showed that Ser83Leu was the most common mutation in gyrA that was observed in all quinolone resistant isolates, followed by Asp87Asn. Ser80Ile mutation in parC was observed in 77.7% of the tested isolates. The prevalence of PMQR genes was 92.5% aac (6)-Ib, 51.8% qnrB, 40.7% qepA, and 37% qnrS.Conclusion: Quinolone resistance is common in K. pneumoniae isolates in Baghdad. The frequent mutation in gyrA and parC, and the presence of PMQR genes is alarming.Keywords: MDR K. pneumoniae, quinolones resistance gene, PMQR

Background: The objective of this study is to evaluate the performance of the Xpert CARBA-R Test and the phenotyping confirmation tests (MHT, CIM, Mastdiscs, and Carba NP) for the detection of carbapenemases in multidrug resistant (MDR) Klebsiella pneumoniae isolates. Methods: A total of 68 MDR K. pneumoniae isolates isolated from various clinical samples, were included in the study. The identification and antibiotic susceptibility tests of these isolates were performed using the VITEK®2 (BioMérieux, France) automated system. The Xpert CARBA-R test was used as the molecular method. The combined disc method was performed using Mastdiscs Combi-D70C that includes four antibiotic discs with specific inhibitors. The modified Hodge test was performed on all isolates. Carbapenemase inactivation method (CIM) and Carba NP test was used for carbapenemase enzyme production. Results: Of the 50 isolates detected to produce carbapenemase by the molecular method (Xpert CARBA-R Test), 45 (90%) were detected by MHT, 39 (78%) were detected by CIM, and 42 (84%) were detected by Mastdiscs, while all the 50 isolates were detected by the Carba NP test. When the Xpert CARBA-R Test was taken as a reference, significant differences were found between the Carba NP and Xpert CARBA-R Test. There was no significant difference between the other phenotypic methods and Xpert CARBA-R Test. The sensitivity of the MHT, CIM, combined disc, and Carba NP tests was calculated as 0.90, 0.78, 0.84, and 1 and their specificity was calculated as 0.83, 0.83, 0.83 and 0, respectively. According to the gold standard, the predictive power of MHT, CIM, and MAST methods was found to be statistically significant. Conclusions: There are various methods of carbapenemase detection, including phenotypic and molecular methods. There is no single detection method that is valid and usable in all conditions. Laboratories should choose a suitable carbapenemase detection and confirmation method in line with their needs, economic conditions, and infrastructures. Although the detection of the presence of carbapenemase by molecular methods is fast and reliable, low-cost phenotypic tests can be used in laboratories that do not have this possibility. It is an important advantage that the combined disc method can also determine the enzyme type. [ABSTRACT FROM AUTHOR]

Background: In recent years enormous use of quinolones leads to increasing resistance towards multiple antimicrobial agents, by various pathogens in different geographical regions with the varying rate. Objective: In this study, we report the antibiotic susceptibility profile and molecular characterization of Quinolones resistant Klebsiella pneumonia isolates originated from Iraq province. Methodology: Klebsiella pneumonia isolates grown from various clinical sources were phenotypically confirmed by a pool of biochemical tests and reconfirmed using gene sequence of rpo housekeeping gene. Antibiotic susceptibility biogram was carried by disc diffusion agar method, quinolones resistant isolates were subjected to ciprofloxacin MIC testing and cartwheel method was used to determine the screening efflux pump activity in resistant isolates. All resistant isolates were subjected to molecular screening to detect resistance genes gyrA, parC, qepA, qnrB, qnrS and aac(6)Ib. Results: Forty-three K. pneumonia isolated were identified by biochemical testing and confirmed by rpo gene sequence. Antibiotic susceptibility results showed a high rate of resistance to Ceftriaxone, gentamicin ciprofloxacin, and Levofloxacin antibiotics. Ciprofloxacin MIC results revealed 96.1% isolates had value above 256 µg/ml. Ciprofloxacin mixed with EtBr at various concentrations resulted in a decrease of MIC. Genetic detection revealed the predominance of gyrA and aac(6)Ib gene. The phylogenetic trees showed the high clonal diversity among isolates. Conclusion: The high percentage of resistance towards different types of antibiotics including fluoroquinolones is considered to be problematic for public health and represents a serious challenge to infectious disease specialists worldwide. [ABSTRACT FROM AUTHOR]

Highlights • SHV-ESBL was detected in E. coli and K. peumoniae clinical isolates. • bla SHV-10 , bla SHV-18 , bla SHV-58 and bla SHV-91 were the most predominant variants. • New point mutations were detected in bla SHV and altered bla SHV enzyme binding sites. • bla SHV gene co-transfer was determined among isolates from human and poultry. • These data are valuable in the field of epidemiology and preventive medicine fields. Abstract The emergence of multidrug-resistant (MDR) pathogens was reported worldwide. Herein, SHV extended-spectrum β-lactamase (SHV-ESBL) variants detection was investigated in MDR E. coli and K. pneumoniae isolates recovered from human subjects (n = 144), one day-old chicks (n = 36) and broiler clinical samples (n = 90). All examined samples were positive for E. coli (n = 246/270; 91.11%) and Klebsiella pneumoniae (n = 24/270; 8.89%). Antimicrobial susceptibility testing was performed on E. coli and K. pneumoniae. SHV-ESBL producing isolates were defined followed by SHV-ESBL amino acids sequence and proteins structure-function analyses. Phylogenetic analysis of 11 MDR isolates resistant to at least 6 β-lactams was designed to determine their genetic relationship with those previously identified in Egypt. SHV-ESBL variants were detected in 28% and 16% of E. coli and K. pneumoniae isolates, respectively. Among the 11 SHV-ESBL producing isolates, one isolate displayed 100% bla SHV-12 similarity with three point mutations, while the other 10 isolates displayed amino acid substitutions at previously non-reported sites. Amino acid sequence analyses of these 10 isolates displayed 96–100% identity to bla SHV-10 (2 isolates with 3–6 point mutations), bla SHV-18 (one isolate with 4 point mutations), bla SHV-58 (4 isolates with 4–5 point mutations), and bla SHV-91 (3 isolates with 3–7 point mutations). These mutations altered SHV-enzyme pocket dimensions and its binding sites chargeability. The bla SHV phylogeny analysis revealed occurrence of variants in closely related lineages with bla SHV-5 and bla SHV-12 with possibility of bla SHV gene transfer between human and birds. The occurrence of these variants in Egypt could help in epidemiological studies and could explain the emergent resistance to β-lactams. [ABSTRACT FROM AUTHOR]

The emergence and spread of pathogens harboring extended spectrum beta-lactamase (ESBL) like carbapenem resistant Gram negative bacteria are the major emerging threat to public health. Of particular concern Klebsiella pneumoniae carbapenamase- producing strains have been recorded worldwide. Catheter associated urinary tract infections (CAUTI) caused by K. pneumoniae are significantly associated with morbidity and mortality. Hence the present work was aimed to develop a strategy for addressing these issues through an innovative approach of antibiofilm and immunomodulation. These two independent activities were analyzed in a Streptomyces derived ASK2 bioactive compound. While analysing the effect of sub-minimum inhibitory concentrations (sub-MICs), 0.5x of Minimum Inhibitory Concentration (MIC) was found to be more effective in preventing biofilm formation on coverslip and silicone catheter. The minimum biofilm eradication concentration (MBEC) was found to be 15-fold higher MIC with eradication of 75% of 3 day old biofilm. Apart from its antibiofilm potential, ASK2 also acts as an opsonin and enhances phagocytic response of macrophages against multidrug resistant K. pneumoniae. In addition, ASK2 resulted in elevated levels of nitric oxide generation by the macrophages and has a stimulating effect on IL-12, IFN-γ, and TNF-α proinflammatory cytokines. The opsonic role of ASK2 and its potential in modulating proinflammatory cytokines secreted by macrophages implies the importance of ASK2 in modulating cellular immune response of macrophages against MDR K. pneumoniae. The present study proposes ASK2 as a promising candidate for treating MDR K. pneumoniae infections with its dual properties of antibiofilm and immunomodulatory activities.