Combating multidrug-resistance in S. pneumoniae: a G-quadruplex binding inhibitor of efflux pump and its bio-orthogonal assembly.

Antibiotic resistance poses a significant global health threat, necessitating innovative strategies to combat multidrug-resistant bacterial infections. Streptococcus pneumoniae, a pathogen responsible for various infections, harbors highly conserved DNA quadruplexes in genes linked to its pathogenesis. In this study, we introduce a novel approach to counter antibiotic resistance by stabilizing G-quadruplex structures within the open reading frames of key resistance-associated genes (pmrA, recD and hsdS). We synthesized An4, a bis-anthracene derivative, using Cu(I)-catalyzed azide-alkyne cycloaddition, which exhibited remarkable binding and stabilization of the G-quadruplex in the pmrA gene responsible for drug efflux. An4 effectively permeated multidrug-resistant S. pneumoniae strains, leading to a substantial 12.5-fold reduction in ciprofloxacin resistance. Furthermore, An4 downregulated pmrA gene expression, enhancing drug retention within bacterial cells. Remarkably, the pmrA G-quadruplex cloned into the pET28a(+) plasmid transformed into Escherichia coli BL21 cells can template Cu-free bio-orthogonal synthesis of An4 from its corresponding alkyne and azide fragments. This study presents a pioneering strategy to combat antibiotic resistance by genetically reducing drug efflux pump expression through G-quadruplex stabilization, offering promising avenues for addressing antibiotic resistance.

Whole genome sequence-based molecular characterization of blood isolates of carbapenem-resistant Enterobacter cloacae complex from ICU patients in Kolkata, India, during 2017-2022: emergence of phylogenetically heterogeneous Enterobacter hormaechei subsp. xiangfangensis.

Blood-borne infections caused by the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) are major public threats with respect to the challenges encountered during treatment. This study describes the whole genome sequencing-based molecular characteristics of blood isolates (n = 70) of CR-ECC from patients admitted to the intensive care unit of tertiary care hospitals in Kolkata, India, during 2017-2022 with respect to species identification, antimicrobial resistance (AMR) profiling, mechanism of drug resistance, and molecular subtypes. Vitek2 MALDI and species-specific PCR identified Enterobacter hormaechei subsp. xiangfangensis (47.14%) as the emerging CR-ECC subspecies in Kolkata. The predominating carbapenemase and extended-spectrum ß-lactamase genes found were blaNDM-1 (51.42%) and blaCTX-M-15 (27%), respectively. Besides, blaNDM-4, blaNDM-5, blaNDM-7, blaCMH-3, blaSFO-1, blaOXA-181, blaOXA-232, blaKPC-3, and blaDHA-7 genes were also detected, which were not previously reported from India. A multitude of Class 1 integrons (including In180, In4874, In4887, and In4888, which were novel) and plasmid replicon types (IncFIB, IncFII, IncX3, IncHI1-HI2, IncC, and IncR) involved in AMR dissemination were identified. Reverse transcription-PCR and western blot revealed that carbapenem resistance in non-carbapenemase-producing CR-ECC isolates was contributed by elevated levels of ampC, overexpression of acrAB, and loss of ompF. A total of 30 distinct sequence types (STs) were ascertained by multi-locus sequence typing; of which, ST2011, ST2018, ST2055, ST2721, and ST2722 were novel STs. Pulsed-field gel electrophoresis analysis showed heterogeneity (69 pulsotypes with a similarity coefficient of 48.40%) among the circulating isolates, suggesting multiple reservoirs of infections in humans. Phylogenetically and genetically diverse CR-ECC with multiple AMR mechanisms mandates close monitoring of nosocomial infections caused by these isolates to forestall the transmission and dissemination of AMR.IMPORTANCEThe emergence and extensive dissemination of the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) have positioned it as a critical nosocomial global pathogen. The dearth of a comprehensive molecular study pertaining to CR-ECC necessitated this study, which is the first of its kind from India. Characterization of blood isolates of CR-ECC over the last 6 years revealed Enterobacter hormaechei subsp. xiangfangensis as the most prevalent subsp., exhibiting resistance to almost all antibiotics currently in use and harboring diverse transmissible carbapenemase genes. Besides the predominating blaNDM-1 and blaCTX-M-15, we document diverse carbapenemase and AmpC genes, such as blaNDM-4, blaNDM-7, blaOXA-181, blaOXA-232, blaKPC-3, blaCMH-3, blaSFO-1, and blaDHA-7, in CR-ECC, which were not previously reported from India. Furthermore, novel integrons and sequence types were identified. Our findings emphasize the need for strengthened vigilance for molecular epidemiological surveillance of CR-ECC due to the presence of epidemic clones with a phylogenetically diverse and wide array of antimicrobial resistance genes in vulnerable populations.

The impact of COVID-19 on health care-associated infections in intensive care units in low- and middle-income countries: International Nosocomial Infection Control Consortium (INICC) findings.

BACKGROUND: This study examines the impact of the COVID-19 pandemic on health care-associated infection (HAI) incidence in low- and middle-income countries (LMICs). METHODS: Patients from 7 LMICs were followed up during hospital intensive care unit (ICU) stays from January 2019 to May 2020. HAI rates were calculated using the International Nosocomial Infection Control Consortium (INICC) Surveillance Online System applying the Centers for Disease Control and Prevention's National Healthcare Safety Network (CDC-NHSN) criteria. Pre-COVID-19 rates for 2019 were compared with COVID-19 era rates for 2020 for central line-associated bloodstream infections (CLABSIs), catheter-associated urinary tract infections (CAUTIs), ventilator-associated events (VAEs), mortality, and length of stay (LOS). RESULTS: A total of 7,775 patients were followed up for 49,506 bed days. The 2019 to 2020 rate comparisons were 2.54 and 4.73 CLABSIs per 1,000 central line days (risk ratio [RR] = 1.85, p = .0006), 9.71 and 12.58 VAEs per 1,000 mechanical ventilator days (RR = 1.29, p = .10), and 1.64 and 1.43 CAUTIs per 1,000 urinary catheter days (RR = 1.14; p = .69). Mortality rates were 15.2% and 23.2% for 2019 and 2020 (RR = 1.42; p < .0001), respectively. Mean LOS for 2019 and 2020 were 6.02 and 7.54 days (RR = 1.21, p < .0001), respectively. DISCUSSION: This study documents an increase in HAI rates in 7 LMICs during the first 5 months of the COVID-19 pandemic and highlights the need to reprioritize and return to conventional infection prevention practices.

Emergence of ceftriaxone resistant Salmonella enterica serovar Typhi in Eastern India.

In view of widespread isolation of fluoroquinolone (FQ) resistant Salmonella enterica serovar Typhi globally, third generation cephalosporins (ceftriaxone) are used as alternative drugs for treatment of typhoid fever in recent years. But reports on emergence of third generation cephalosporin resistant S. Typhi have been documented from various countries including India posing threat in future use of this drug for typhoid treatment. Here, we report on genomic analysis of a third generation cephalosporin resistant S. Typhi strain isolated for the first time from Eastern India, Kolkata during 2019. The study strain was phenotypically resistant to ceftriaxone, ampicillin. Whole genome sequencing revealed the presence of conjugative IncX3 plasmid carrying blaSHV-12 gene on it. The study strain belongs to H58 haplotype (4.3.1.2) and ST1 type. Comparison of phylogenetic analysis of the study strain with other cephalosporin resistant S. Typhi strains across the world revealed that three strains isolated from Western part of India during 2016 were its closest neighbours. Hence close monitoring of cephalosporin resistant S. Typhi strains are of great importance to control the furure use of this antibiotic for the treatment of typhoid fever.

Changes in antimicrobial resistance and molecular attributes of Salmonellae causing enteric fever in Kolkata, India, 2014-2018.

Globally, enteric fever caused by Salmonella Typhi (S. Typhi, ST) and S. Paratyphi A (SPA) remain one of the major diseases of public health importance. In this study, a total of 457 (380 ST, 77 SPA) blood isolates were collected from three tertiary care hospitals in Kolkata during 2014-18. Additionally, 66 (3.4%) ST and 5 (0.25%) SPA were recovered from blood culture of 1962 patients attending OPD of one pediatric hospital during 2016-18. The study isolates were tested for antimicrobial resistance (AMR) profiles; AMR genes; molecular sub-types by PFGE, MLVA and CRISPR. Among the total 446 ST and 82 SPA isolates, fluoroquinolone (FQ) resistance was very common in both serovars. Ciprofloxacin resistance of 24.9% and 9.8% & ofloxacin resistance of 20.9% and 87.8% were found in ST and SPA respectively. Majority (>70%) of the isolates showed decreased susceptibility to ciprofloxacin (DCS). A single point mutation in gyrA gene (S83F) was responsible for causing DCS in 37.5% (n = 42/112) ST and 63% (n = 46/73) SPA isolates. Multidrug resistance (MDR) was found only in 3.4% ST isolates and encoded the genes blaTEM-1, catA, sul, strA-strB, class 1 integron with dfrA7. All MDR ST (n = 15) possessed non-conjugative non-IncHI1 (180 kb) plasmid except one having conjugative IncHI1 (230 kb) plasmid and one without plasmid. The MDR genes were integrated near chromosomal cyaA gene site in ST with/without the presence of plasmid (nonIncH1). Almost 65.7% resistant ST belonged to H58 haplotype. PFGE showed clonally related isolates with 81% similarity in ST and 87% in SPA. Similarly, CRISPR typing showed less diversity among the isolates. However, the isolates (ST and SPA) were found to be more diverse by MLVA typing (D value 0.987 and 0.938). The study reports decrease in MDR and increase in FQ resistance among typhoidal Salmonella isolates over the years giving interesting information for enteric fever treatment.

Cytidine-Derived Hydrogels with Tunable Antibacterial Activities.

We herein report the preparation of hydrogels by the Ag+ ion induced assembly of cytidine and boronic acids. These hydrogels, presumably formed by an i-motif like arrangement of cytidine and its boronate ester analogues, possess excellent thixotropic and self-healing properties. The hydrogels exhibit potent antimicrobial activity that can be tuned by varying the functional groups in their boronic acid component. These hydrogels could find potential use as antimicrobial agents and stimuli-responsive drug delivery systems.