First Record of the Isolation of blaNDM-1-Producing Enterotoxigenic Enterobacter cloacae ssp. cloacae in Baghdad/Iraq.

BACKGROUND: Antibiotic resistance is a major problem threatening human beings. The genetic determinants that carry resistance genes can be transmitted in several ways in clinical and food environments. Hence, this research study aimed to investigate the presence of New Delhi metallo-beta-lactamase-1 (blaNDM-1) produced by enterotoxigenic Enterobacter cloacae in both clinical and food samples. METHODS AND RESULTS: Twenty-four isolates of Enterobacter spp. were isolated, seven isolates from food samples and 17 isolates from blood taken from neonates and children (1 day - 10 years old) resident in a children's hospital. Antibiotic susceptibility test to 14 antibiotics was performed for all isolates. Enterotoxigenicity of the clinical and foodborne isolates was detected phenotypically using Suckling mouse bioassay. Genomic deoxyribonucleic acid (DNA) was extracted from the isolated Enterobacter spp. that were detected resistant to imipenem. Polymerase chain reaction (PCR) was used to amplify blaNDM-1 gene followed by sequencing. The results of the bioassay revealed that 64.28% of E. cloacae ssp. cloacae isolates were enterotoxigenic. Two E. cloacae ssp. cloacae were imipenem resistant. CONCLUSIONS: This study showed that one isolate from a male child 1 < year was bla NDM-1 positive that was con-firmed by sequencing. This is the first report that revealed blaNDM-1 producing Enterobacter cloacae in Iraq.

Phenotypic and genetic characterization of a lipolytic Ralstonia mannitolilytica isolate from petroleum-contaminated soil in Iraq.

BACKGROUND: Lipases play a crucial role in various industrial applications, and microbial lipases, particularly those from bacteria, possess significant properties. With increasing concerns about the environmental and health impacts of hydrocarbons from pipelines and refineries, there is a growing need to mitigate the risks associated with these compounds. METHODS: In this study, 40 bacterial isolates were recovered from contaminated soil samples collected from multiple refineries across Iraq. Using the Vitek system, bacterial isolates were identified up to the species level, revealing that only 12 isolates exhibited lipase-producing capabilities. RESULTS: Among the lipase-producing isolates, Ralstonia mannitolilytica demonstrated the highest extracellular lipase activity, as determined by an olive oil plate assay supplemented with rhodamine B. Confirmation of the species identity was achieved through 16S rRNA gene sequencing, with the obtained sequence deposited under accession number LC772176.1. Further sequence analysis revealed single nucleotide polymorphisms (SNPs) in the genome of Ralstonia mannitolilytica strain H230303-10_N19_7x_R2 (CP011257.1, positions 1,311,102 and 1,311,457). Additionally, the presence of the lipase gene was confirmed through amplification and sequencing using a thermocycler PCR. Sequence analysis of the gene, aligned using Geneious Prime software, identified SNPs (CP010799, CP049132, AY364601, CP011257, and CP023537), and a phylogenetic tree was constructed based on genetic characterization. CONCLUSION: Our findings highlight the potential of Ralstonia mannitolilytica as a promising candidate for lipase production and contribute to our understanding of its genetic diversity and biotechnological applications in hydrocarbon degradation and industrial processes.

The Correlation between Adhesion Genes and Biofilm Formation among Escherichia coli Clinical Isolates.

BACKGROUND: The adhesion genes are responsible for biofilm production which leads to chronic diseases like urinary tract infections (UTIs). Uropathogenic Escherichia coli (UPEC) is the most predominant pathogen involved in UTIs. This study aims to evaluate the relationship between adhesion genes and bacterial biofilm that form by UPEC. METHODS: Fifty clinical isolates of E. coli from patients infected with UTIs were identified and antimicrobial resistance was tested by MIC assay. A polymerase chain reaction (PCR), a quick and sensitive assay to identify the adhesions operon (Afa, papG, flu, and fimH), was developed using eight primers and used for amplification. E. coli K-12 strain and E. coli J96 were used as a negative and a positive control for detection of adhesion genes. RESULTS: The study reported 70% of isolates produce strong biofilm. Adhesion genes showed as follow Afa (64% n = 33), papG (42% n = 23), flu (94% n = 52), fimH (86% n = 45). CONCLUSIONS: The resistance to non-Beta lactam antibiotic was significantly correlated with the availability of genes that encode for adhesion. These genes were highly correlated to biofilm formation in E. coli clinical isolates.

Neonatal intensive care units: extended spectrum ß-lactamase genes and biofilm formation by Serratia marcescens.

BACKGROUND: The increasing cases of bloodstream infections among children at neonatal intensive care units (NICUs) led this work to investigate biofilm production, antibiotics and the presence of ESßL genes in Serratia marcescens (S. marcescens) strains isolated from blood. METHODS: Twenty S. marcescens strains were isolated and identified by the VITEK-2 system over 7 months from late 2022 to mid-2023 from Ibn Al-Balady Hospital in Baghdad. Kirby-Bauer test was used to measure antibiotic susceptibility. RESULTS: The results revealed that 95% of twenty S. marcescens isolates were non-susceptible to Ampicillin and Amoxicillin-clavulanic. Furthermore, S. marcescens isolates showed a high sensitivity rate 70% toward Imipenem. All S. marcescens strains 100% were produced biofilm. This work clarifies that, out of 20 S. marcescens strains, 80% were harbored ESßL genes. The coexistence of blaTEM, blaCTX and blaSHV genes was shown in 43.75% of strains, while 56.25% of S. marcescens strains harbored single ES[Formula: see text]L genes. The biofilm values increase with the accuracy of EsßL genes. Phylogenetic analyses based on the sequence of blaCTX-M and blaTEM were done with closely related genes in the GenBank using MEGA6 software. CONCLUSIONS: The distribution of blaTEM, blaCTX and blaSHV genes among local S. marcescens strains may be attributed to the indiscriminate use of antibiotics. The results confirmed the spread of ESßL genes in S. marcescens from blood infections among newborn infants.

New Phage cocktail against infantile Sepsis bacteria.

A total of 54 positive blood aerobic cultures of suspected blood sepsis in neonates were purified, diagnosed and tested for antibiotic susceptibility. Six bacterial pathogens of Escherichia coli (10 isolates), Klebsiella pneumoniae (10 isolates), Haemophilus influenzae (4 isolates), Pseudomonas aeruginosa (3 isolates), Citrobacter fruendii (1 isolate) and Moraxella catarrhalis (1 isolate) were selected for preparation of phages active against all isolates of each species. Virulent phages towards bacterial isolates were isolated from sewage water by spot lysis method, and a total of 29 phages active towards selected bacterial pathogens were isolated and purified. Phage(s) active against each bacterial species showed activity spectrum within each species of 30-80%, 50-80%, 50%, 50%, 100% and 100% for isolates of Escherichia coli, Klebsiella spp., Haemophilus influenzae, Pseudomonas aeruginosa, Citrobacter fruendii and Moraxella catarrhalis, respectively. A total of 29 phages were formulated in one cocktail in a concentration of 106 PFU/ml in SM buffer for each of a total of 29 phages in SM buffer and showed activity spectrum of 100% against all their bacterial hosts in vitro.

An experimental mouse model for phage therapy of bacterial pathogens causing bacteremia.

A minimum lethal dose on mice for five bacterial pathogens were identified with PCR of neonatal septicemia of Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Citrobacter freundii and Moraxella catarrhalis were studied. Bacteriophage preparations active against 10 isolates of E. coli, 10 isolates of K. pneumoniae, 4 isolates of H. influenzae, 3 isolates of P. aeruginosa, 1 isolate of C. freundii and 1 isolate of M. catarrhalis were tested for their efficacy in treatment of experimental mouse bacteremia of those isolates through dose-ranging and delayed-treatment studies. Efficacy of polyvalent phage preparations of different phages belonging to same species against pathogens of the same species was studied. Results showed that monovalent and polyvalent phage preparations were active in treatment of mice bacteremia after 45 min of injection of bacterial pathogens and on intervals of 5 h, 8 h, 14 h, 18 h and 24 h after bacterial pathogen injection.

Treatment strategy by lactoperoxidase and lactoferrin combination: Immunomodulatory and antibacterial activity against multidrug-resistant Acinetobacter baumannii.

Lactoperoxidase (Lpo) and Lactoferrin (Lf) were extracted from camel colostrum milk and purified. The antibacterial activity of the two purified proteins was estimated against 14 isolates of multidrug resistance Acinetobacter baumannii. A combination of Lpo and Lf exhibited bactericidal action against A. baumannii in vitro. A mouse model of acute A. baumannii pneumonia was improved. The injection of combined Lpo and Lf after infection leads to significant clearance of A. baumannii rates in lung as well as blood culture P < 0.05 in comparing with control. Furthermore, the results showed a significant P < 0.05 reduction in the Bronchoalveolar lavage albumin concentration, lung injury and lactate dehydrogenase activity in comparing with control. In addition, the combination of Lpo and Lf treatment induced substantial elevation of IL-4 and IL10 concentrations p < 0.0 5 that helped to prevent damage caused by the inflammatory response. We concluded that combination of Lpo and Lf had a major inhibition effect against A. baumannii in comparing with imipenem as well as their immunomodulatory activity against resistant A. baumannii was increased by a synergistic effect of them as a crude combination. This study indicated two combined proteins consider as crucial strategy for practical treatment of pneumonia in the future.

Two novel roles of buffalo milk lactoperoxidase, antibiofilm agent and immunomodulator against multidrug resistant Salmonella enterica serovar Typhi and Listeria monocytogenes.

The increasing occurrence of multidrug resistant bacteria causing bacteremia infection, constitutes a major health problem, difficult-to-treat bacteremia due to its ability to form biofilm. Buffalo milk lactoperoxidase (BMLpo) is effective and safe to use as bacteriostatic agent. The MIC of BMLpo and amikacin were used to evaluate the antibiofilm activity against resistant L. monocytogenes and S. typhi. Prophylactic effects of BMLpo against L. monocytogenes and S. typhi bacteremia in vivo have been tested and ELISA test used to evaluate serum cytokines. Significant antibiofilm activity of BMLpo observed against the highest biofilm producer isolates. Our results showed that the prophylactic effect of BMLpo in BALB/c mice bacteremic model. A significant clearance of L. monocytogenes and S. typhi, investigated in blood and different organs tissues in BMLpo-treated infected groups when compared to the non-treated groups. Further, analysis of serum cytokines levels revealed that BMLpo prophylaxis modulates their release in different way when it compared to the control. This study showed, BMLpo effects as an alternative antibiofilm agent to compact gram negative pathogens, and protects the host against bacteremia infection. Moreover, the BMLpo role as an immunomodulatory. These investigations indicated the BMLpo crucial role in the practical clinical applications.

Investigating the link between imipenem resistance and biofilm formation by Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC ≤ 2 µg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilm-deficient mutants became imipenem resistant (MIC ≥ 8 µg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.