Potassium-induced partial inhibition of lactoperoxidase: structure of the complex of lactoperoxidase with potassium ion at 2.20 Å resolution.

Lactoperoxidase, a heme-containing glycoprotein, catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite which acts as an antibacterial agent. The prosthetic heme moiety is attached to the protein through two ester linkages via Glu258 and Asp108. In lactoperoxidase, the substrate-binding site is formed on the distal heme side. To study the effect of physiologically important potassium ion on the structure and function of lactoperoxidase, the fresh protein samples were isolated from yak (Bos grunniens) colostrum and purified to homogeneity. The biochemical studies with potassium fluoride showed a significant reduction in the catalytic activity. Lactoperoxidase was crystallized using 200 mM ammonium nitrate and 20% PEG-3350 at pH 6.0. The crystals of LPO were soaked in the solution of potassium fluoride and used for the X-ray intensity data collection. Structure determination at 2.20 Å resolution revealed the presence of a potassium ion in the distal heme cavity. Structure determination further revealed that the propionic chain attached to pyrrole ring C of the heme moiety, was disordered into two components each having an occupancy of 0.5. One component occupied a position similar to the normally observed position of propionic chain while the second component was found in the distal heme cavity. The potassium ion in the distal heme cavity formed five coordinate bonds with two oxygen atoms of propionic moiety, Nε2 atom of His109 and two oxygen atoms of water molecules. The presence of potassium ion in the distal heme cavity hampered the catalytic activity of lactoperoxidase.

Prevalence, clinical & biochemical correlates of non-alcoholic fatty liver disease in overweight adolescents.

BACKGROUND & OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) characterized by excessive accumulation of fat in the liver, which can progress to inflammation, and cirrhosis, has emerged as an important complication of obesity in adults as well as children. This study was undertaken to assess the prevalence of NAFLD and its correlation with clinical and biochemical parameters in overweight Indian adolescents. METHODS: In this cross-sectional study, 218 overweight adolescents aged 10 to 16 yr and their parents were included. Measurements included anthropometry, ultrasonography to diagnose NAFLD, fasting glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lipids for adolescents and parents, and additional parameters of blood pressure, body fat percentage (BF%), fasting insulin, apolipoprotein C3, tumour necrosis factor-α and adiponectin for adolescents. The variables were compared between adolescents with and without NAFLD, and logistic regression analysis was performed. RESULTS: Mean age and body mass index (BMI)SD score (SDS) were 11.9±1.6 yr and 2.3±1.1, respectively. NAFLD was seen in 62.5 per cent of the adolescents. The prevalence of NAFLD in the parents was similar among the adolescents with and without NAFLD, while BMI and waist circumference SDS, BF per cent, blood pressure (BP), ALT, AST, insulin and homeostatic model assessment of insulin resistance (HOMA-IR) were significantly higher in the adolescents with NAFLD. On multiple logistic regression, abdominal obesity, HOMA-IR and BF per cent were independently associated with NAFLD with odds ratios (95% confidence interval) of 2.77 (1.40-5.47), 2.21 (1.16-4.21) and 2.17 (1.12-4.22), respectively. INTERPRETATION & CONCLUSIONS: NAFLD was noted among nearly two-thirds of the overweight adolescents. An independent association was observed between abdominal obesity, HOMA-IR and body fat percentage and NAFLD in overweight adolescents.

Structural evidence of the conversion of nitric oxide (NO) to nitrite ion (NO2-) by lactoperoxidase (LPO): Structure of the complex of LPO with NO2- at 1.89 Å resolution.

Lactoperoxidase (LPO) is a heme containing mammalian enzyme which uses hydrogen peroxide (H2O2) to catalyze the conversion of substrates into oxidized products. LPO is found in body fluids and tissues such as milk, saliva, tears, mucosa and other body secretions. The previous structural studies have shown that LPO converts substrates, thiocyanate (SCN-) and iodide (I-) ions into oxidized products, hypothiocyanite (OSCN-) and hypoiodite (IO-) ions respectively. We report here a new structure of the complex of LPO with an oxidized product, nitrite (NO2-). This product was generated from NO using the two step reaction of LPO by adding hydrogen peroxide (H2O2) in the solution of LPO in 0.1 M phosphate buffer at pH 6.8 as the first step. In the second step, NO gas was added to the above mixture. This was crystallized using 20% (w/v) PEG-3350 and 0.2 M ammonium iodide at pH 6.8. The structure determination showed the presence of NO2- ion in the distal heme cavity of the substrate binding site of LPO. The structure also showed that the propionate group which is linked to pyrrole ring D of the heme moiety was disordered. Similarly, the side chain of Asp108, which is covalently linked to heme moiety, was also split into two components. As a result of these changes, the conformation of the side chain of Arg255 was altered allowing it to form new interactions with the disordered carboxylic group of propionate moiety. These structural changes are indicative of an intermediate state in the catalytic reaction pathway of LPO.

Molecular Screening of Carbapenem-Resistant K. pneumoniae (CRKP) Clinical Isolates for Concomitant Occurrence of Beta-Lactam Genes (CTX-M, TEM, and SHV) in the Kingdom of Bahrain.

The emergence of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae, including CRKP infections, has resulted in significant morbidity and mortality worldwide. We aimed to explore the presence of bla genes (CTX-M, TEM, and SHV) in CRKP isolates. A total of 24 CRKP isolates were randomly selected from the Salmaniya Medical Complex Microbiology Laboratory. These isolates, which were positive for carbapenemases, were further explored for CTX-M, TEM, and SHV genes using PCR. All the CTX-M PCR amplicons were sent for sequencing. To determine genetic relatedness, molecular typing by ERIC-PCR was performed. The bla gene testing demonstrated that a significant proportion of these isolates harbored SHV, CTX-M, and TEM genes (100%, 91.6%, and 45.8%), respectively. Bioinformatic analyses confirmed CTX-M-15 in these isolates. ERIC-PCR analysis showed three clusters demonstrating genetic relatedness. The study findings reveal the concomitant carriage of the SHV and CTX-M-15 and a comparatively lower carriage of TEM genes in CRKP isolates. Our findings highlight the significance of routinely reporting the presence of antibiotic resistance genes along with regular antibiotic sensitivity reports, as this will aid clinicians in prescribing appropriate antibiotics.

Evolution and implementation of One Health to control the dissemination of antibiotic-resistant bacteria and resistance genes: A review.

Antibiotic resistance is a serious threat to humanity and its environment. Aberrant usage of antibiotics in the human, animal, and environmental sectors, as well as the dissemination of resistant bacteria and resistance genes among these sectors and globally, are all contributing factors. In humans, antibiotics are generally used to treat infections and prevent illnesses. Antibiotic usage in food-producing animals has lately emerged as a major public health concern. These medicines are currently being utilized to prevent and treat infectious diseases and also for its growth-promoting qualities. These methods have resulted in the induction and spread of antibiotic resistant infections from animals to humans. Antibiotics can be introduced into the environment from a variety of sources, including human wastes, veterinary wastes, and livestock husbandry waste. The soil has been recognized as a reservoir of ABR genes, not only because of the presence of a wide and varied range of bacteria capable of producing natural antibiotics but also for the usage of natural manure on crop fields, which may contain ABR genes or antibiotics. Fears about the human health hazards of ABR related to environmental antibiotic residues include the possible threat of modifying the human microbiota and promoting the rise and selection of resistant bacteria, and the possible danger of generating a selection pressure on the environmental microflora resulting in environmental antibiotic resistance. Because of the connectivity of these sectors, antibiotic use, antibiotic residue persistence, and the existence of antibiotic-resistant bacteria in human-animal-environment habitats are all linked to the One Health triangle. The pillars of support including rigorous ABR surveillance among different sectors individually and in combination, and at national and international level, overcoming laboratory resource challenges, and core plan and action execution should be strictly implemented to combat and contain ABR under one health approach. Implementing One Health could help to avoid the emergence and dissemination of antibiotic resistance while also promoting a healthier One World. This review aims to emphasize antibiotic resistance and its regulatory approaches from the perspective of One Health by highlighting the interconnectedness and multi-sectoral nature of the human, animal, and environmental health or ill-health facets.

Proteomic Analysis of the Colistin-resistant E. coli Clinical Isolate: Explorations of the Resistome.

BACKGROUND: Antimicrobial resistance is a worldwide problem after the emergence of colistin resistance since it was the last option left to treat carbapenemase-resistant bacterial infections. The mcr gene and its variants are one of the causes for colistin resistance. Besides mcr genes, some other intrinsic genes are also involved in colistin resistance but still need to be explored. OBJECTIVE: The aim of this study was to investigate differential proteins expression of colistin-resistant E. coli clinical isolate and to understand their interactive partners as future drug targets. METHODS: In this study, we have employed the whole proteome analysis through LC-MS/MS. The advance proteomics tools were used to find differentially expressed proteins in the colistin-resistant Escherichia coli clinical isolate compared to susceptible isolate. Gene ontology and STRING were used for functional annotation and protein-protein interaction networks, respectively. RESULTS: LC-MS/MS analysis showed overexpression of 47 proteins and underexpression of 74 proteins in colistin-resistant E. coli. These proteins belong to DNA replication, transcription and translational process; defense and stress related proteins; proteins of phosphoenol pyruvate phosphotransferase system (PTS) and sugar metabolism. Functional annotation and protein-protein interaction showed translational and cellular metabolic process, sugar metabolism and metabolite interconversion. CONCLUSION: We conclude that these protein targets and their pathways might be used to develop novel therapeutics against colistin-resistant infections. These proteins could unveil the mechanism of colistin resistance.

Structural evidence of the oxidation of iodide ion into hyper-reactive hypoiodite ion by mammalian heme lactoperoxidase.

Lactoperoxidase (1.11.1.7, LPO) is a mammalian heme peroxidase found in the extracellular fluids of mammals including plasma, saliva, airway epithelial lining fluids, nasal lining fluid, milk, tears, gastric juices, and intestinal mucosa. To perform its innate immune action against invading microbes, LPO utilizes hydrogen peroxide (H2 O2 ) to convert thiocyanate (SCN- ) and iodide (I- ) ions into the oxidizing compounds hypothiocyanite (OSCN- ) and hypoiodite (IO- ). Previously determined structures of the complexes of LPO with SCN- , OSCN- , and I- show that SCN- and I- occupy appropriate positions in the distal heme cavity as substrates while OSCN- binds in the distal heme cavity as a product inhibitor. We report here the structure of the complex of LPO with IO- as the first structural evidence of the conversion of iodide into hypoiodite by LPO. To obtain this complex, a solution of LPO was first incubated with H2 O2 , then mixed with ammonium iodide solution and the complex crystallized by the addition of PEG-3350, 20% (wt/vol). These crystals were used for X-ray intensity data collection and structure analysis. The structure determination revealed the presence of four hypoiodite ions in the substrate binding channel of LPO. In addition to these, six other hypoiodite ions were observed at different exterior sites. We surmise that the presence of hypoiodite ions in the distal heme cavity blocks the substrate binding site and inhibits catalysis. This was confirmed by activity experiments with the colorimetric substrate, ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-sulfonic acid)), in the presence of hypoiodite and iodide ions.

Clinical carbapenem-resistant Klebsiella pneumoniae isolates simultaneously harboring bla NDM-1, bla OXA types and qnrS genes from the Kingdom of Bahrain: Resistance profile and genetic environment.

The prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) is currently increasing worldwide, prompting WHO to classify it as an urgent public health threat. CRKP is considered a difficult to treat organism owing to limited therapeutic options. In this study, a total of 24 CRKP clinical isolates were randomly collected from Salmaniya Medical Complex, Bahrain. Bacterial identification and antibiotic susceptibility testing were performed, on MALDI-TOF and VITEK-2 compact, respectively. The isolates were screened for carbapenem resistance markers (bla NDM, bla OXA-23, bla OXA-48 and bla OXA-51) and plasmid-mediated quinolone resistance genes (qnrA, qnrB, and qnrS) by monoplex PCR. On the other hand, only colistin-resistant isolates (n=12) were screened for MCR-1, MCR-2 and MCR-3 genes by monoplex PCR. Moreover, the Genetic environment of bla NDM, integrons analysis, and molecular characterization of plasmids was also performed. Antibiotic susceptibility revealed that all the isolates (100%) were resistant to ceftolozane/tazobactam, piperacillin/tazobactam, 96% resistant to ceftazidime, trimethoprim/sulfamethoxazole, 92% resistant to meropenem, gentamicin and cefepime, 88% resistant to ciprofloxacin, imipenem, and 37% resistant to amikacin. Ceftazidime/avibactam showed the least resistance (12%). 75% (n=12/16) were resistant to colistin and 44% (n=7/16) showed intermediate susceptibility to tigecycline. The detection of resistant determinants showed that the majority (95.8%) of CRKP harbored bla NDM-1, followed by bla OXA-48 (91.6%) bla OXA-51 (45.8%), and bla OXA-23 (41.6%). Sequencing of the bla NDM amplicons revealed the presence of bla NDM-1. Alarmingly, 100% of isolates showed the presence of qnrS. These predominant genes were distributed in various combinations wherein the majority were bla NDM-1 + bla OXA-51+ qnrS + bla OXA-48 (n =10, 41.7%), bla NDM-1 + bla OXA-23+ qnrS + bla OXA-48 (n=8, 33.3%), among others. In conclusion, the resistance rate to most antibiotics is very high in our region, including colistin and tigecycline, and the genetic environment of CRKP is complex with the carriage of multiple resistance markers. Resistance to ceftazidime/avibactam is uncommon and hence can be used as a valuable option for empirical therapy. Molecular data on resistance markers and the genetic environment of CRKP is lacking from this geographical region; this would be the first report addressing the subject matter. Surveillance and strict infection control strategies should be reinforced in clinical settings to curb the emergence and spread of such isolates.

Structure of Yak Lactoperoxidase at 1.55 Å Resolution.

Lactoperoxidase (LPO) is a heme containing oxido-reductase enzyme. It is secreted from mammary, salivary, lachrymal and mucosal glands. It catalyses the conversion of thiocyanate into hypothiocyanate and halides into hypohalides. LPO belongs to the superfamily of mammalian heme peroxidases which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The heme prosthetic group is covalently linked in LPO through two ester bonds involving conserved residues Glu258 and Asp108. It was isolated from colostrum of yak (Bos grunniens), purified to homogeneity and crystallized using ammonium iodide as a precipitating agent. The crystals belonged to monoclinic space group P21 with cell dimensions of a = 53.91 Å, b = 78.98 Å, c = 67.82 Å and ß = 92.96°. The structure was determined at 1.55 Å resolution. This is the first structure of LPO from yak. Also, this is the highest resolution structure of LPO determined so far from any source. The structure determination revealed that three segments (Ser1-Cys15), (Thr117-Asn138) and (Cys167-Leu175) were disordered and formed one surface of LPO structure. In the substrate binding site, the iodide ions were observed in three subsites which are formed by (1) heme moiety and residues, Gln105, Asp108, His109, Phe113, Arg255, Glu258, Phe380 and Phe381, (2) residues, Asn230, Lys232, Pro236, Cys248, Phe254, Phe381 and Pro424 and (3) residues, Ser198, Leu199 and Arg202. The structure determination also revealed that the side chain of Phe254 was disordered. It was observed to adopt two conformations in the structures of LPO.

Co-existence of blaNDM-1 and blaVIM-1 producing Moellerella wisconsensis in NICU of North Indian Hospital.

Infections caused by carbapenemase-producing Enterobacteriaceae have become a major threat to public health, worldwide. Here we report clinically significant NDM-1 and VIM-1 producing Moellerella wisconsensis which has not yet been described in the literature; this is the first report of M. wisconsensis strain harbouring blaNDM-1 and blaVIM-1, recovered from the rectal swab of a low birth weight female child admitted in NICU of the north Indian tertiary care hospital. A plasmid of IncW incompatibility with size of 154 kb was observed in AK-92 strain.

Outbreak of Efficiently Transferred Carbapenem-Resistant blaNDM-Producing Gram-Negative Bacilli Isolated from Neonatal Intensive Care Unit of an Indian Hospital.

The emergence of blaNDM particularly in Gram-negative bacteria is a burden on the health care system in developing countries. Hence, this study was initiated to screen New Delhi Metallo-ß-lactamase (NDM)-producing Gram-negative bacterial strains from neonatal intensive care unit (NICU) of an Indian Hospital. A total of 18 blaNDM-producing isolates were detected in the present study. Out of 18 blaNDM variant isolates, 6 were Klebsiella pneumoniae, 4 Escherichia coli, 2 Enterobacter aerogenes, 1 Acinetobacter lwoffii, 1 Enterobacter cloacae, 3 Acinobacter baumannii, and 1 Cedecea davisae from NICU, showing resistance against all antibiotics, except colistin and polymixin. The transferability of resistance determinants was tested by conjugation. Transfer of blaNDM-producing strains was successful in all 18 strains. In the case of transconjugants, the minimum inhibitory concentration values were found to decrease. The blaNDM-producing isolates contained detectable plasmids of size 66, 38, and 6 kb. Plasmi/d-based replicon typing revealed the incompatibility types Inc (A/C, FIIA, FIC, K, F, W, FIA, P, X, FIB, B/O) in blaNDM-carrying isolates. This study revealed the outbreak of multiple variants of blaNDM (13 NDM-1, 4 NDM-5, and 1 NDM-7). Moreover, other resistance markers, viz. blaOXA-1, blaCMY-1, blaVIM-1, and blaSHV-1 coassociated with blaNDM were also found. In this study, we reported NDM-producing C. davisae as a first report to the best of our knowledge. This study is an attempt to reveal the dissemination of blaNDM isolated from neonates in NICU and their efficient transferability among Gram-negative bacilli through horizontal gene transfer.

Molecular characterization of novel sequence type of carbapenem-resistant New Delhi metallo-ß-lactamase-1-producing Klebsiella pneumoniae in the neonatal intensive care unit of an Indian hospital.

Emergence of multi-drug resistance, especially carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major threat to public health. The aim of this study was to characterize CRKP isolates from infants admitted to the Neonatal Intensive Care Unit (NICU) to find the clonal outbreak of New Delhi metallo-ß-lactamase (NDM) producers. In this study 17 CRKP isolates were analysed. Antimicrobial susceptibility of the isolates was determined by the disc diffusion and micro-dilution method. Carba-NP test and double-disk synergy test (DDST) were performed for the detection of carbapenemase and metallo-ß-lactamase-producing K. pneumoniae. Antibiotic-resistant markers were detected by polymerase chain reaction (PCR) followed by sequencing. Clonal relatedness of the isolates was checked by multi-locus sequence typing. Conjugation experiments were performed to determine the transferability of the plasmids. All 17 CKRP isolates were found to carry blaNDM (13 blaNDM-1, 1 blaNDM-4 and 3 blaNDM-5), seven isolates carried blaOXA-48, 13 isolates had blaCTX-M-15, seven isolates carried blaCMY-1 and five isolates were found to carry blaSHV-1 on conjugative plasmids of different types (IncFIA, IncFIB, IncFIIAs, IncFIC, IncA/C, IncF, IncK, IncX, IncW and IncY). Of six different sequence types (STs) identified, ST3344 was detected as a novel ST in two K. pneumoniae isolates. Genetic environment analysis revealed ISAba125 and bleomycin genes flanking to all blaNDM variants. This is the first report of novel ST3344 in two NDM-1-producing K. pneumoniae isolates from neonates admitted to the NICU of a North Indian Hospital. This study is provides understanding of the genetic features of this newly emerged strain type.

Genetic polymorphisms associated with obesity and non-alcoholic fatty liver disease in Asian Indian adolescents.

Background The objective of this study was to investigate the association of polymorphisms in four genes, tumor necrosis factor-α (TNFA), patatin-like phospholipase domain containing 3 (PNPLA3), adiponectin (ADIPOQ) and apolipoprotein C3 (APOC3), with obesity and non-alcoholic fatty liver disease (NAFLD) in Asian Indian adolescents. Methods In this case-control study, 218 Asian Indian adolescents with overweight/obesity and 86 lean healthy adults without fatty liver were enrolled. Hepatic steatosis was assessed and graded by ultrasonography (USG). Serum insulin, lipids, alanine aminotransferase (ALT), aspartate aminotransferase (AST), TNF-α, adiponectin and apolipoprotein C3 were measured and genotyping was done. Frequencies of variant and wild genotypes in all adolescents and in the subgroups without steatosis, with grade 1 steatosis and with grade 2 or 3 steatosis were compared to those in the controls. The frequencies were also compared in the overweight adolescents with grade 2 or 3 steatosis and without steatosis. Results Variant genotypes of polymorphisms -863 C > A and -1031 T > C of the TNFA gene, 455 T > C of the APOC3 gene and the wild type of +276 G > T of the ADIPOQ gene were associated with obesity with odds ratios (OR, 95% confidence interval [CI]) of 2.5 (1.5-4.4), 2.5 (1.5-4.2), 2.0 (1.1-3.6) and 2.5 (1.4-5.0), respectively. Polymorphisms 455 T > C of APOC3 and rs738409 C > G of PNPLA3 were associated with NAFLD. Fasting insulin and triglycerides (TG) were higher in the adolescents with homozygous variant polymorphisms -1031 T > C of TNFA and 455 T > C of APOC3 genes, respectively. Conclusions Several polymorphisms were noted to have a significant association with obesity and NAFLD in Asian Indian adolescents.

Detection of New Delhi Metallo-ß-Lactamase Variants NDM-4, NDM-5, and NDM-7 in Enterobacter aerogenes Isolated from a Neonatal Intensive Care Unit of a North India Hospital: A First Report.

A total 402 Enterobacteriaceae isolates were recovered from blood and rectal swabs of 1,000 infants admitted to the Neonatal Intensive Care Unit (NICU) of the Jawaharlal Medical College and Hospital Aligarh, India. Carbapenamase producers were determined by Carba NP phenotype biochemical assay. Out of 402 isolates, it was the first time three of the isolates were identified as Enterobacter aerogenes carrying blaNDM-4, blaNDM-5, and blaNDM-7 genes. These genes were identified by polymerase chain reaction (PCR) and sequence analysis. The isolates were further characterized to know the plasmid type and genetic environment features, including integron and IS elements. All the three E. aerogenes isolates (AK-93, AK-95, and AK-96) were resistant to all ß-lactams, including carbapenems. The ß-lactamase genes blaOXA-1, blaOXA-9, blaSHV-1, and blaVIM-2 were also found to be coassociated with blaNDM-4 in AK-93, blaOXA-1, blaOXA-9, and blaCMY-149 were found to be coexisted with blaNDM-5 in AK-95, and blaOXA-1; blaOXA-9, and blaCMY-145 were also found to be coassociated with blaNDM-7 in AK-96, identified by PCR analysis. Plasmid-based replicon typing revealed plasmids of different incompatibility in E. aerogenes in each of the isolates, AK-93 AK-95, and AK-96, respectively. ERIC-PCR was performed for the analysis of genetic relatedness of the strains. We found blaNDM-4, blaNDM-5, and blaNDM-7 producing three E. aerogenes strains, which were not clonally related. Genetic environment analysis revealed the presence of bleomycin resistance gene (bleMBL) to downstream of blaNDM and complete ISAba125 sequence were found upstream of blaNDM in all the three variants of these isolates. This is the first time we have identified blaNDM-4, blaNDM-5, and blaNDM-7 in E. aerogenes species, isolated from the NICU of a tertiary care hospital in India.

Occurrence of blaNDM Variants Among Enterobacteriaceae From a Neonatal Intensive Care Unit in a Northern India Hospital.

Carbapenem-resistance among enterobacteriaceae has become a global health concern. The objective of this study was to understand NDM producing enterobacteriaceae and their genetic basis of resistance, spreading in neonatal intensive care unit. Carbapenem resistant NDM producing enterobacteriaceae isolates were recovered from rectal swab and blood sample of infants admitted in NICU. These were determined by using Carba-NP test. All isolates were identified using BD PhoenixTM-100 and MICs were determined by broth microdilution method. The blaNDM and associated resistant markers were checked by PCR followed by sequencing. Moreover, ERIC-PCR and genetic environment of blaNDM gene were also performed for the analysis of clonal relationship and genetic surrounding of the strains. We characterized 44 isolates with blaNDM variants in Escherichia coli (45.5%), Klebsiella pneumoniae (40.9%), Citrobacter freundii (4.5%), Citrobacter braakii (2.3%), Klebsiella oxytoca (2.3%), Enterobacter cloacae (2.3%), Enterobacter aerogenes (2.2%) from NICU, showing resistance against all antibiotics except colistin and polymixin B. ISAba125 and bleomycin gene were found surrounding all blaNDM variants, besides class I integron on plasmid. (ERIC)-PCR data revealed non-clonal relatedness among most of the isolates. The transfer of resistant markers was confirmed by conjugation experiment. The PCR-based replicon typing was carried out using DNA of transconjugants. These isolates carried NDM-1 (20.45%), NDM-4 (36.36%), NDM-5 (38.64%), NDM-7 (4.55%), along with OXA, CMY, and SHV variants on conjugative plasmid of IncFIA, IncFIC, IncF, IncK, IncFIB, IncB/O, IncHI1, IncP, IncY, IncFIIA, IncI1, and IncN types. An increased number of carbapenem-resistant NDM producing enterobacteriaceae isolates recovered from NICU which is alarming signal for health workers and policy makers. Hence, it is utmost important to think about infection control measures.

CRISPRi Induced Suppression of Fimbriae Gene (fimH) of a Uropathogenic Escherichia coli: An Approach to Inhibit Microbial Biofilms.

Urinary tract infection (UTI) is one the common infections caused by the recalcitrant nature of biofilms, developed after the pathogen has adhered to the inner lining of the urinary tract. Although significant research has been made in recent years to control these types of infection, but as of yet, no approach has sufficiently been able to reduce the prevalence of UTIs. The main objective of this study was to prevent UTIs through targeting the fimH gene, which is the major virulent factor responsible for biofilm formation. The novelty of this work lies in the use of CRISPRi, a gene specific editing tool to control such types of infections. Accordingly, the system was designed to target fimH gene, responsible for bacterial adherence and this approach was successfully validated by performing microscopic, biofilm and adherence assays.