Bacterial abundance and antimicrobial resistance patterns of uropathogens among pregnant women with asymptomatic bacteriuria: Association with glycemic status.

Objectives: Antimicrobial resistance (AMR), a growing global menace, poses a significant threat to maternal and fetal health. Gestational diabetes mellitus (GDM) causes double trouble in pregnancy, increasing the risk of a variety of infectious morbidities while also raising the possible association with AMR. Asymptomatic bacteriuria (ASB) is a common problem in pregnancy, but little research has been done to date explicitly examining the relationship between GDM and ASB and yielded conflicting results. Even fewer studies have specifically examined the relationship between GDM and AMR in women with ASB. Retrieving the most recent information on the disease burden, the range of causative pathogens, their patterns of AMR, and associated risk factors in pregnant women is crucial to stop the exponential rise in AMR in pregnancy and improve maternal and neonatal outcomes of infectious morbidities. Hence, this study was planned to investigate the association between glycemic status and the contemporary bacterial profile, antimicrobial resistance(AMR), and associated variables among pregnant women with ASB. Study design: This prospective, hospital-based, cross-sectional study was conducted among 320 pregnant women; divided into two groups, GDM and non-GDM. Data regarding sociodemographic and clinical characteristics were collected using a structured questionnaire. Clean-catch midstream urine samples were investigated for the presence of significant bacterial uropathogens and their AMR pattern was determined using recommended culture methods. Results: We found ASB in 46.25% of study participants with significantly higher occurrence in the GDM group. Dominant isolates were Escherichia coli followed by Klebsiella pneumoniae. AMR was noted in 51.35% and multidrug resistance(MDR) in 23.65% of isolates. Overall AMR, MDR and higher degrees of AMR were higher among uropathogens isolated from the GDM group as compared to the non GDM group, although the difference was not statistically significant. Conclusion: The high occurrence of ASB in pregnancy along with substantially high AMR in this study suggests the need for effective infection control and stewardship programmes. By defining the association of ASB and AMR with hyperglycemia, our study calls for the exploitation of this potential association in halting the pandemic of AMR and in improving the management of infectious morbidities, thus in-turn alleviating their undesired maternal and infant outcomes.

Efficacy of Novel Combinations of Antibiotics against Multidrug-Resistant-New Delhi Metallo-Beta-Lactamase-Producing Strains of Enterobacterales.

The emergence of multidrug-resistance (MDR)-New Delhi metallo-beta-lactamase (NDM)-producing microorganisms-has become a serious concern for treating such infections. Therefore, we investigated the effective antimicrobial combinations against multidrug-resistant New Delhi metallo-beta-lactamase-producing strains of Enterobacterales. The tests were carried out using the 2D(two-dimensional) checkerboard method. Of 7 antimicrobials, i.e., doripenem (DRP), streptomycin (STR), cefoxitin (FOX), imipenem (IPM), cefotaxime (CTX), meropenem (MER), and gentamicin (GEN), 19 different combinations were used, and out of them, three combinations showed synergistic effects against 31 highly drug-resistant strains carrying blaNDM and other associated resistance markers. Changes in the minimum inhibitory concentration (MIC) values were interpreted using the test fractional inhibitory concentration index (FIC Index). The FIC Index values of these combinations were found in the range of 0.1562 to 0.5, which shows synergy, whereas no synergism was observed in the remaining antimicrobial combinations. We conclude that these antibiotic combinations can be analyzed in in vivo and pharmacological studies to establish an effective therapeutic approach.

Moist Heat Synthesis of Magnetic EGCG-Cappedα-Fe2O3 Nanoparticles and Their In Vitro and In Silico Interactions with Pristine HSA- and NDM-1-Producing Bacteria.

A simple, facile, moist-heating (e.g., autoclave), one-step procedure for EGCG-mediated biosynthesis of narrow-size magnetic iron oxide (α-Fe2O3) nanoparticles (EGCG-MINPs) was developed. The influence of pH of the reaction mixture over the size distribution of as-synthesized EGCG-MINPs was investigated systematically by employing UV-visible (UV-vis) spectroscopy and dynamic light scattering (DLS)-based hydrodynamic size, surface charge (zeta-potential), and polydispersity index (PDI). The FE-SEM, TEM, and XRD characterizations revealed that the EGCG-MINPs synthesized at pH 5.0 were in the size range of 6.20-16.7 nm and possess well-crystalline hexagonal shaped nanostructures of hematite (α-Fe2O3) crystal phase. The role of EGCG in Fe3+ ion reduction and EGCG-MINP formation was confirmed by FTIR analysis. The VSM analysis has revealed that EGCG-MINPs were highly magnetic nanostructures with the hysteretic feature of saturation magnetization (Ms), remanent magnetization (Mr), and coercivity (Hc) as 33.64 emu/g, 12.18 emu/g, and 0.33 Oe, respectively. Besides, significant (p < 0.001) dose-dependent (250-1000 µg/mL) antibacterial and antibiofilm activities against the NDM-1-producing Gram-negative Escherichia coli (AK-33), Klebsiella pneumoniae (AK-65), Pseudomonas aeruginosa (AK-66), and Shigella boydii (AK-67) bacterial isolates warranted the as-synthesized EGCG-MINPs as a promising alternative for clinical management of chronic bacterial infections in biomedical settings. In addition, molecular docking experiments revealed that compared to free Fe3+ and EGCG alone, the EGCG-MINPs or Fe-EGCG complex possess significantly high binding affinity toward HSA and hence can be considered as promising biocompatible nanodrug carriers in in vivo drug delivery systems.

Association of vaginal dysbiosis and gestational diabetes mellitus with adverse perinatal outcomes.

OBJECTIVE: Gestational diabetes mellitus (GDM) is associated with adverse perinatal outcomes and is an independent risk factor for vaginal dysbiosis. Understanding the vaginal microbiota in health and disease is essential to screen, detect, and manage complications of pregnancy. Therefore, the aims of the present study were to assess and compare vaginal dysbiosis in pregnancy in women with and without GDM and examine its impact on perinatal outcomes in our population. METHODS: The present study was a prospective cohort study recruiting pregnant women. The subjects were divided into two groups (GDM and non-GDM) and were followed until delivery to assess fetomaternal outcomes. Vaginal samples were collected at 24-28 weeks and 34-38 weeks for Nugent scoring and determination of bacterial and fungal species. RESULTS: The study recruited 502 pregnant women, with a final assessment of 320 mother-infant pairs (GDM n = 134; non-GDM n = 186). We found a significant association of vaginal dysbiosis with GDM and adverse perinatal outcomes. Significant differences were also seen in status of infection and its trimester-wise changes in relation to hyperglycemia. CONCLUSION: By defining an association of vaginal dysbiosis with GDM and its correlation with perinatal outcomes, the present study calls for exploitation of this potential association as a new target in the prevention and treatment of GDM and in alleviating their undesired maternal and infant outcomes.

A mechanistic approach to prove the efficacy of combination therapy against New Delhi metallo-ß-lactamases producing bacterial strain: a molecular and biochemical approach.

BACKGROUND: NDM-1 is a novel broad-spectrum metallo-ß-lactamase with the capability to grant resistance to almost all ß-lactam antibiotics. Its widespread dissemination made treatment options a major challenge to combat, causing threat to public health worldwide. Due to antibiotic resistance problems, development of effective therapeutics for infections caused by NDM-1 producing strains is urgently required. Since combination therapies are proved to be effective in many cases, this study was initiated to put forward novel effective antibiotics combinations for fighting infections caused by NDM-1 producing strains. METHODS: Streptomycin and amikacin combination and streptomycin and ciprofloxacin combination were tested by checkerboard assay. NDM-1 protein/enzyme was then expressed and purified to carry out enzyme kinetics study, CD and fluorescence spectroscopic studies. RESULTS: Streptomycin and amikacin combination and streptomycin and ciprofloxacin combination showed synergistic effect towards NDM-1 producing bacterial strains as shown by FICI results. NDM-1 producing bacterial cells were expressed and purified to obtain protein as the source of enzyme. When NDM-1 enzyme was treated with streptomycin along with amikacin, the efficiency of enzyme was decreased by 49.37% and when the enzyme was treated with streptomycin along with ciprofloxacin, the efficiency of enzyme was decreased by 29.66% as revealed by enzyme kinetic studies. Due to binding of streptomycin and amikacin in combination and streptomycin and ciprofloxacin in combination, conformational changes in the secondary structure of NDM-1 enzyme were observed by CD spectroscopic studies. Antibiotics streptomycin and ciprofloxacin bind with NDM-1 through exothermic processes, whereas amikacin binds through an endothermic process. All three antibiotics bind spontaneously with an association constant of the order of 104 M-1 as revealed by fluorescence spectroscopic studies. CONCLUSIONS: The therapeutic combination of streptomycin with amikacin and ciprofloxacin plays an important role in inhibiting NDM-1 producing bacterial strains. Therefore, these combinations can be used as effective future therapeutic candidates against NDM-1 producing bacterial cells.

Whole-genome sequence analysis of multidrug-resistant Staphylococcus epidermidis ST35 strain isolated from human ear infection of an Iraqi patient.

OBJECTIVES: Staphylococcus epidermidis is the most common coagulase-negative Staphylococcus colonising the human skin and mucous membranes and is a major cause of nosocomial infections. Antimicrobial resistance (AMR) in S. epidermidis has increased significantly in the last few decades, threatening human health globally. METHODS: This study explored the AMR status in an S. epidermidis isolate (AK-612) from an ear infection of an Iraqi student who had undergone treatment for the same. Whole-genome sequence analysis was also performed. RESULTS: Strain AK-612 is a methicillin-resistant S. epidermidis (MRSE) isolate possessing an SCCmec type V element. Strain AK-612 belongs to sequence type 35 (ST35), clonal complex 2 (CC2), which to the best of our knowledge has not been reported previously in Iraq, having been previously reported only in Portugal and Germany. In Portugal, S. epidermidis ST35 was reported to colonise the nasal area of a bird of prey (Buteo buteo) that undergoes a migratory period extending its range from Europe to Asia. The plasmid of S. epidermidis AK-612 showed greatest identity with plasmid 1 of S. epidermidis strain PM221, which was isolated in Finland from an intramammary bovine infection. This ST35 strain may have crossed continental boundaries and expanded its occurrence in animals and humans. CONCLUSION: This is a matter of serious concern as the dissemination of multidrug-resistant S. epidermidis in human infections is a major hindrance for the treatment of these infections. Transmission of this isolate across continental boundaries will make infection control a difficult task.

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.

Synergistic effect of doripenem in combination with cefoxitin and tetracycline in inhibiting NDM-1 producing bacteria.

Aim: To propose newer combinations of antibiotics effective against NDM-1-producing bacterial strains. Materials & methods: Antibiotics combinations were tested by checkerboard assay. NDM-1 protein/enzyme was expressed and purified to perform enzyme kinetics, circular dichroism and fluorescence spectroscopic studies. Results: Doripenem-cefoxitin combination and doripenem-tetracycline combination showed synergistic effect toward NDM-1-producing strains. The catalytic efficiency of NDM-1 enzyme was decreased drastically by 96.6% upon doripenem-cefoxitin treatment and by 35.54% after doripenem-tetracycline treatment. Conformational changes were observed in NDM-1 upon combination treatment. Conclusion: NDM-1-producing bacterial strains show resistance to multiple antibiotics but the combination of doripenem-cefoxitin and doripenem-tetracycline are effective against them. The combination of a carbapenem and cephamycin antibiotic is proposed for future treatment options against bacteria-producing NDM-1.

Significant role of Asn-247 and Arg-64 residues in close proximity of the active site in maintaining the catalytic function of CTX-M-15 type ß-lactamase.

Members of Enterobacteriaceae cause antibiotic-resistant infections worldwide. One such marker, CTX-M-15, expressed by Enterobacteriaceae produces ß-lactamases, which hydrolyze the cephalosporin group of antibiotics, such as cefotaxime, used in the treatment of both Gram-positive and negative bacterial infections. Amino acid residues present in close proximity of the active site might also play a major role in the structure and function of CTX-M-15, hence the objective of this study was to investigate the significance of two amino acid residues, Asn-247 and Arg-64, present near to the active site in the hydrolysis of cefotaxime. bla CTX-M-15, cloned from the E. cloacae strain, and using Polymerase Chain Reaction (PCR)-based site-directed mutagenesis, Asn247Val and Arg64Leu mutations were introduced. The minimum inhibitory concentrations of cefotaxime for the CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were reduced by 512 and 128 fold, respectively. Proteins/enzymes of wild-type CTX-M-15, CTX-M-15 (N247V) and CTX-M-15 (R64L) mutants were expressed and purified. Kinetic studies showed that the catalytic efficiencies of the N247V mutant and R64L mutant enzymes in the hydrolysis of cefotaxime were reduced by 89.66% and 71.11%, respectively. Circular dichroism spectroscopic studies showed considerable changes in the α-helical content of the mutant enzymes. A fluorescence study showed that N247V mutant-cefotaxime and R64L mutant-cefotaxime underwent complex formation with strong interactions. The study provides an understanding of the crucial role of the amino acid residues asparagine 247 and arginine 64 present in close proximity of the active site in the hydrolytic mechanism of CTX-M-15 type ß-lactamases. Hence, Asn-247 and Arg-64 can be used as potential target sites for the design of inhibitory molecules against CTX-M-15-producing bacterial strains.

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.