Exploring the resistome, virulome, and mobilome of multidrug-resistant Klebsiella pneumoniae isolates: deciphering the molecular basis of carbapenem resistance.

BACKGROUND: Klebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019-February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples. RESULTS: The draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, blaNDM, and blaOXA, respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins. CONCLUSIONS: This study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources.

A multifunctional vanillin-infused chitosan-PVA hydrogel reinforced by nanocellulose and CuO-Ag nanoparticles as antibacterial wound dressing.

Wound healing is an intricate and ever-evolving phenomenon that involves a series of biological processes and multiple stages. Despite the growing utilization of nanoparticles to enhance wound healing, these approaches often overlook properties like mechanical stability, toxicity, and efficacy. Hence, a multifunctional wound dressing is fabricated using Chitosan-PVA membrane crosslinked with vanillin and reinforced with nano-cellulose and CuO-Ag nanoparticles in this study. FTIR, SEM, and XRD were employed to study the morphology and structural properties of the membrane. Biomedical tests including biodegradability, antimicrobial study, cytotoxicity, and animal models were conducted to evaluate the membrane's performance as a wound healing material. The membrane displayed impressive mechanical strength, measuring as high as 49.985 ± 2.31 MPa, and had a hydrophilic nature, with moisture retention values up to 98.84 % and swelling percentages as high as 191.67 %. It also demonstrated biodegradable properties and high cell viability of up to 92.30 %. Additionally, the fabricated membranes exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria, with maximum zone of inhibition measuring 16.8 ± 0.7 mm and 9.2 ± 0.1 mm, respectively. Moreover, the membranes also demonstrated superior wound healing properties. These results suggested great potential of fabricated membranes as an effective wound dressing material.

Characterization of exclusively non-commensal Neisseria gonorrhoeae pangenome to prioritize globally conserved and thermodynamically stable vaccine candidates using immune-molecular dynamic simulations.

Neisseria gonorrhoeae (Ngo) has emerged as a global threat leading to one of the most common sexually transmitted diseases in the world. It has also become one of the leading antimicrobial resistant organisms, resulting in fewer treatment options and an increased morbidity. Therefore, in recent years, there has been an increased focus on the development of new treatments and preventive strategies to combat its infection. In this study, we have combined the most conserved epitopes from the completely assembled strains of Ngo to develop a universal and a thermodynamically stable vaccine candidate. For our vaccine design, the epitopes were selected for their high immunogenicity, non-allergenicity and non-cytotoxicity, making them the ideal candidates for vaccine development. For the screening process, several reverse vaccinology tools were employed to rigorously extract non-homologous and immunogenic epitopes from the selected proteins. Consequently, a total number of 3 B-cell epitopes and 6 T-cell epitopes were selected and joined by multiple immune-modulating adjuvants and linkers to generate a promiscuous immune response. Additionally, the stability and flexible nature of the vaccine construct was confirmed using various molecular dynamic simulation tools. Overall, the vaccine candidate showed promising binding affinity to various HLA alleles and TLR receptors; however, further studies are needed to assess its efficacy in-vivo. In this way, we have designed a multi-subunit vaccine candidate to potentially combat and control the spread of N. gonorrhoeae.

Assesment of polyphenolic compounds against biofilms produced by clinical Acinetobacter baumannii strains using in silico and in vitro models.

Several types of microbial infections are caused by Acinetobacter baumanii that has developed resistance to antimicrobial agents. We therefore investigated the role of plant polyphenols against A. baumannii using in silico and in vitro models. The clinical strains of A. baumannii were investigated for determination of resistance pattern and resistance mechanisms including efflux pump, extended spectrum beta lactamase, phenotype detection of AmpC production, and Metallo-ß-lactamase. The polyphenolic compounds were docked against transcription regulator BfmR (PDB ID 6BR7) and antimicrobial, antibiofilm, and anti-quorum sensing activities were performed. The antibiogram studies showed that all isolated strains were resistant. Strain A77 was positive in Metallo-ß-lactamase production. Similarly, none of strains were producers of AmpC, however, A77, A76, A75 had active efflux pumps. Molecular docking studies confirmed a strong binding affinity of Rutin and Catechin towards transcription regulator 6BR7. A significant antimicrobial activity was recorded in case of quercetin and syringic acid (MIC 3.1 µg/mL) followed by vanillic acid and caffeic acid (MIC 12.5 µg/mL). All tested compounds presented a strong antibiofilm activity against A. baumanii strain A77 (65 to 90%). It was concluded that all tested polyphenols samples posess antimicrobial and antibiofilm activities, and hence they may be utilized to treat multidrug resistance A. baumannii infections.

Intensive care unit sinks are persistently colonized with multidrug resistant bacteria and mobilizable, resistance-conferring plasmids.

Contamination of hospital sinks with microbial pathogens presents a serious potential threat to patients, but our understanding of sink colonization dynamics is largely based on infection outbreaks. Here, we investigate the colonization patterns of multidrug-resistant organisms (MDROs) in intensive care unit sinks and water from two hospitals in the USA and Pakistan collected over 27 months of prospective sampling. Using culture-based methods, we recovered 822 bacterial isolates representing 104 unique species and genomospecies. Genomic analyses revealed long-term colonization by Pseudomonas spp. and Serratia marcescens strains across multiple rooms. Nanopore sequencing uncovered examples of long-term persistence of resistance-conferring plasmids in unrelated hosts. These data indicate that antibiotic resistance (AR) in Pseudomonas spp. is maintained both by strain colonization and horizontal gene transfer (HGT), while HGT maintains AR within Acinetobacter spp. and Enterobacterales, independent of colonization. These results emphasize the importance of proactive, genomic-focused surveillance of built environments to mitigate MDRO spread. IMPORTANCE Hospital sinks are frequently linked to outbreaks of antibiotic-resistant bacteria. Here, we used whole-genome sequencing to track the long-term colonization patterns in intensive care unit (ICU) sinks and water from two hospitals in the USA and Pakistan collected over 27 months of prospective sampling. We analyzed 822 bacterial genomes, representing over 100 different species. We identified long-term contamination by opportunistic pathogens, as well as transient appearance of other common pathogens. We found that bacteria recovered from the ICU had more antibiotic resistance genes (ARGs) in their genomes compared to matched community spaces. We also found that many of these ARGs are harbored on mobilizable plasmids, which were found shared in the genomes of unrelated bacteria. Overall, this study provides an in-depth view of contamination patterns for common nosocomial pathogens and identifies specific targets for surveillance.

High resistance of fluoroquinolone and macrolide reported in avian pathogenic Escherichia coli isolates from the humid subtropical regions of Pakistan.

OBJECTIVES: This study aimed to assess the antimicrobial resistance profile, virulence potential, and genetic characterization of avian pathogenic Escherichia coli (APEC) that cause colibacillosis in poultry. METHODS: Antibiotic susceptibility testing (AST) was measured via the Kirby-Bauer disc diffusion method against 27 commonly used antibiotics. Phylogrouping, virulence-associated gene detection, and hybrid strain detection via multiplex polymerase chain reaction (PCR) and genetic diversity were analysed via ERIC-PCR fingertyping method. RESULTS: AST analysis showed 100% of isolates were multidrug-resistant (MDR) and highest resistance was against penicillin, tetracycline, and macrolide classes of antibiotics. The mcr-1 gene was present in 40% of the isolates, though only 4% of isolates were showing phenotypic resistance. Despite the scarce use of fluoroquinolone, carbapenem, and cephalosporin in the poultry sector, resistance was evident because of the high prevalence of extended-spectrum ß-lactamase (ESBL) (53.7%) and other ß-lactamases in APEC isolates. ß-lactamase genotyping of APEC isolates revealed that 85.7% of isolates contained either blaCTX or blaTEM and around 38% of isolates were complement resistant. Growth in human urine was evident in 67.3% of isolates. Phylogroup B1 (51%) was the most prevalent group followed by phylogroups A (30.6%), D (13.61%), and B2 (4.76%). The most prevalent virulence-associated genes were fimH, iss, and tatT. Results showed that 26 isolates (17.69%) can be termed hybrid strains and APEC/EHEC (enterohemorrhagic E. coli) was the most prevalent hybrid E. coli pathotype. ERIC-PCR fingerprinting genotype analysis clustered APEC isolates in 40 groups (E1-E40). This study provides insights into the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. CONCLUSIONS: The findings of this study provide insights into that the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. This data can inform future studies designed to better estimate the severity of the colibacillosis in poultry farms.

Navigating the brain: the role of exosomal shuttles in precision therapeutics.

Brain diseases have become one of the leading roots of mortality and disability worldwide, contributing a significant part of the disease burden on healthcare systems. The blood-brain barrier (BBB) is a primary physical and biological obstacle that allows only small molecules to pass through it. Its selective permeability is a significant challenge in delivering therapeutics into the brain for treating brain dysfunction. It is estimated that only 2% of the new central nervous system (CNS) therapeutic compounds can cross the BBB and achieve their therapeutic targets. Scientists are exploring various approaches to develop effective cargo delivery vehicles to promote better therapeutics targeting the brain with minimal off-target side effects. Despite different synthetic carriers, one of the natural brain cargo delivery systems, "exosomes," are now employed to transport drugs through the BBB. Exosomes are naturally occurring small extracellular vesicles (EVs) with unique advantages as a therapeutic delivery system for treating brain disorders. They have beneficial innate aspects of biocompatibility, higher stability, ability to cross BBB, low cytotoxicity, low immunogenicity, homing potential, targeted delivery, and reducing off-site target effects. In this review, we will discuss the limitations of synthetic carriers and the utilization of naturally occurring exosomes as brain-targeted cargo delivery vehicles and highlight the methods for modifying exosome surfaces and drug loading into exosomes. We will also enlist neurodegenerative disorders targeted with genetically modified exosomes for their treatment.

Co-occurrence of antibiotic resistance and virulence Genes in Methicillin Resistant Staphylococcus aureus (MRSA) Isolates from Pakistan.

Introduction: Methicillin resistant Staphylococcus aureus (MRSA) is one of the major human pathogen that is associated with hospital as well as community acquired infections and is responsible for huge amount of life-threatening diseases. Objective: Objective of the study was to determine MRSA prevalence, their antibiotic sensitivity patterns, frequency of virulence genes (sea, seb, sed, tst, hla, hld) and their co-occurrence with resistance marker mecA among Rawalpindi and its nearby regions of Pakistani clinical isolates. Methodology: The present study was carried out to identify the virulence and antibiotic resistance genes that co-occur in MRSA through polymerase chain reaction. Antibiotic sensitivity, presence of virulence genes and their co-occurrence with resistance marker mecA were analyzed. Results: These isolates were found resistant to number of antibiotics i.e. Amoxicillin (16.1%), Cefixime (48.38%), Doxycycline (27.415), Trimethoprim/sulfamethoxazole (37.09%), Clindamycin (30.64%), Erythromycin (83.87%), Penicillin (100%), Vancomycin (4.83%), Ciprofloxacin (70.96%), Tetracycline (20%), Linezolid (3.22%) and Fusidic acid (11.295). The frequency of antibiotic resistant gene (mecA) was 69.35% and that of virulence genes hla, hld, sea, seb, sed and tst was 100, 100, 53.2, 30.6, 3.2 and 24.2% respectively. Amongst all examined genes, hla and hld genes had the highest and sed gene had the lowest frequency. The maximum coexistence of genes was observed for hla+hld+mecA gene combination (42 out of 62 isolates). Conclusion: This study reports the presence of multidrug resistant, vancomycin-resistant and mecA negative MRSA isolates in infected patients of Rawalpindi and nearby regions of Pakistan that may have attributed to treatment failures, adaptability of new virulence characteristics and spread of antibiotic resistance.

Genomic Surveillance of Clinical Pseudomonas aeruginosa Isolates Reveals an Additive Effect of Carbapenemase Production on Carbapenem Resistance.

Carbapenem resistance in Pseudomonas aeruginosa is increasing globally, and surveillance to define the mechanisms of such resistance in low- and middle-income countries is limited. This study establishes the genotypic mechanisms of ß-lactam resistance by whole-genome sequencing (WGS) in 142 P. aeruginosa clinical isolates recovered from three hospitals in Islamabad and Rawalpindi, Pakistan between 2016 and 2017. Isolates were subjected to antimicrobial susceptibility testing (AST) by Kirby-Bauer disk diffusion, and their genomes were assembled from Illumina sequencing data. ß-lactam resistance was high, with 46% of isolates resistant to piperacillin-tazobactam, 42% to cefepime, 48% to ceftolozane-tazobactam, and 65% to at least one carbapenem. Twenty-two percent of isolates were resistant to all ß-lactams tested. WGS revealed that carbapenem resistance was associated with the acquisition of metallo-ß-lactamases (MBLs) or extended-spectrum ß-lactamases (ESBLs) in the blaGES, blaVIM, and blaNDM families, and mutations in the porin gene oprD. These resistance determinants were found in globally distributed lineages, including ST235 and ST664, as well as multiple novel STs which have been described in a separate investigation. Analysis of AST results revealed that acquisition of MBLs/ESBLs on top of porin mutations had an additive effect on imipenem resistance, suggesting that there is a selective benefit for clinical isolates to encode multiple resistance determinants to the same drugs. The strong association of these resistance determinants with phylogenetic background displays the utility of WGS for monitoring carbapenem resistance in P. aeruginosa, while the presence of these determinants throughout the phylogenetic tree shows that knowledge of the local epidemiology is crucial for guiding potential treatment of multidrug-resistant P. aeruginosa infections. IMPORTANCE Pseudomonas aeruginosa is associated with serious infections, and treatment can be challenging. Because of this, carbapenems and ß-lactam/ß-lactamase inhibitor combinations have become critical tools in treating multidrug-resistant (MDR) P. aeruginosa infections, but increasing resistance threatens their efficacy. Here, we used WGS to study the genotypic and phylogenomic patterns of 142 P. aeruginosa isolates from the Potohar region of Pakistan. We sequenced both MDR and antimicrobial susceptible isolates and found that while genotypic and phenotypic patterns of antibiotic resistance correlated with phylogenomic background, populations of MDR P. aeruginosa were found in all major phylogroups. We also found that isolates possessing multiple resistance mechanisms had significantly higher levels of imipenem resistance compared to the isolates with a single resistance mechanism. This study demonstrates the utility of WGS for monitoring patterns of antibiotic resistance in P. aeruginosa and potentially guiding treatment choices based on the local spread of ß-lactamase genes.

Evaluation of Blood-Brain-Barrier Permeability, Neurotoxicity, and Potential Cognitive Impairment by Pseudomonas aeruginosa's Virulence Factor Pyocyanin.

Pyocyanin (PCN) is a redox-active secondary metabolite produced by Pseudomonas aeruginosa as its primary virulence factor. Several studies have reported the cytotoxic potential of PCN and its role during infection establishment and progression. Considering its ability to diffuse through biological membranes, it is hypothesized that PCN can gain entry into the brain and induce oxidative stress across the blood-brain barrier (BBB), ultimately contributing towards reactive oxygen species (ROS) mediated neurodegeneration. Potential roles of PCN in the central nervous system (CNS) have never been evaluated, hence the study aimed to evaluate PCN's probable penetration into CNS through blood-brain barrier (BBB) using both in silico and in vivo (Balb/c mice) approaches and the impact of ROS generation via commonly used tests: Morris water maze test, novel object recognition, elevated plus maze test, and tail suspension test. Furthermore, evidence for ROS generation in the brain was assessed using glutathione S-transferase assay. PCN demonstrated BBB permeability albeit in minute quantities. A significant hike was observed in ROS generation (P < 0.0001) along with changes in behavior indicating PCN permeability across BBB and potentially affecting cognitive functions. This is the first study exploring the potential role of PCN in influencing the cognitive functions of test animals.

Methicillin resistant Staphylococcus aureus: A brief review of virulence and resistance.

Staphylococcus aureus is a common gram-positive human pathogen involved in both community-acquired and nosocomial infections ranging from localised superficial lesions to food poisoning and fatal systemic infections owing to its impressive array of virulence factors responsible for attaching, colonising, invading, and avoiding host immune system. The discovery of antibiotics effectively checked the once deadly infections. However, resistance started soon after their discovery and the first methicillin-resistant strain of staphylococcus aureus was reported in the early 1960s. The most important attribute of methicillin-resistant staphylococcus aureus is its acquisition of mecA gene coding for penicillin-binding protein-2a that blocks inhibitory action on peptidoglycan cross-linking. Methicillin-resistant staphylococcus aureus presents a serious global healthcare concern being responsible for prolonged hospital stays and increased mortality. The precise information of virulence factors and resistant traits of methicillin-resistant staphylococcus aureus and their interplay in a community is key to minimize the intermixing of resistant and susceptible pathogens in the community.

In-Silico Vaccine Design Based on a Novel Vaccine Candidate Against Infections Caused by Acinetobacter baumannii.

Acinetobacter baumannii is notorious for causing serious infections of the skin, lungs, soft tissues, bloodstream, and urinary tract. Despite the overwhelming information available so far, there has still been no approved vaccine in the market to prevent these infections. Therefore, this study focuses on developing a rational vaccine design using the technique of epitope mapping to curb the infections caused by A. baumannii. An outer membrane protein with immunogenic potential as well as all the properties of a good vaccine candidate was selected and used to calculate epitopes for selection on the basis of a low percentile rank, high binding scores, good immunological properties, and non-allergenicity. Thus, a 240 amino-acid vaccine sequence was obtained by manually joining all the epitopes in sequence-wise manner with the appropriate linkers, namely AAY, GPGPG, and EAAAK. Additionally, a 50S ribosomal protein L7/L12, agonist to the human innate immune receptors was attached to the N-terminus to increase the overall immune response towards the vaccine. As a result, enhanced overall protein stability, expression, immunostimulatory capabilities, and solubility of the designed construct were observed. Molecular dynamic simulations revealed the compactness and stability of the polypeptide construct. Moreover, molecular docking exhibited strong binding of the designed vaccine with TLR-4 and TLR-9. In-silico immune simulations indicated an immense increment in T-cell and B-cell populations. Bioinformatic tools also significantly assisted with optimizing codons which allowed for successful cloning of constructs into desired host vectors. Using in-silico tools to design a vaccine against A. baumannii demonstrated that this construct could pave the way for successfully combating infections caused by multidrug-resistant bacteria. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-021-10316-7.

Antimicrobial Resistance and Genomic Characterization of Six New Sequence Types in Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates from Pakistan.

Pseudomonas aeruginosa (P. aeruginosa) is a major bacterial pathogen associated with a variety of infections with high mortality rates. Most of the clinical P. aeruginosa isolates belong to a limited number of genetic subgroups characterized by multiple housekeeping genes' sequences (usually 5-7) through the Multi-Locus Sequence Typing (MLST) scheme. The emergence and dissemination of novel multidrug-resistant (MDR) sequence types (ST) in P. aeruginosa pose serious clinical concerns. We performed whole-genome sequencing on a cohort (n = 160) of MDR P. aeruginosa isolates collected from a tertiary care hospital lab in Pakistan and found six isolates belonging to six unique MLST allelic profiles. The genomes were submitted to the PubMLST database and new ST numbers (ST3493, ST3494, ST3472, ST3489, ST3491, and ST3492) were assigned to the respective allele combinations. MLST and core-genome-based phylogenetic analysis confirmed the divergence of these isolates and positioned them in separate branches. Analysis of the resistome of the new STs isolates revealed the presence of genes blaOXA-50, blaPAO, blaPDC, blaVIM-2, aph(3')-IIb, aac(6')-II, aac(3)-Id, fosA, catB7, dfrB2, crpP, merP and a number of missense and frame-shift mutations in chromosomal genes conferring resistance to various antipseudomonal antibiotics. The exoS, exoT, pvdE, rhlI, rhlR, lasA, lasB, lasI, and lasR genes were the most prevalent virulence-related genes among the new ST isolates. The different genotypic features revealed the adaptation of these new clones to a variety of infections by various mutations in genes affecting antimicrobial resistance, quorum sensing and biofilm formation. Close monitoring of these antibiotic-resistant pathogens and surveillance mechanisms needs to be adopted to reduce their spread to the healthcare facilities of Pakistan. We believe that these strains can be used as reference strains for future comparative analysis of isolates belonging to the same STs.

Prevalence of Bacteria and Antimicrobial Resistance Genes in Hospital Water and Surfaces.

Purpose Antimicrobial resistance (AMR) has become a worldwide environmental and public health problem, causing more than 250,000 deaths per year. Unregulated usage, unsafe hospital practices, and misuse in veterinary contribute to the development of multidrug resistance in various bacteria. Hospital water was hypothesized to be a hotspot for AMR transmission because of (1) increased exposure to antibiotic load, (2) poor drainage and sanitation system, (3) interaction between environmental and clinical microbes. The purpose of the research was to assess the biodiversity and AMR in hospital tap waters. Methodology In this study, the microflora of the hospital tap water and hospital surfaces was observed by obtaining water samples from the intensive care unit (ICU), surgical wards, and washrooms. These were processed through membrane filtration and spread on seven different media (Aeromonas Medium, Azide Dextrose Agar, MacConkey Agar, Mannitol Salt Agar, Pseudomonas Cetrimide Agar, Salmonella Shigella Agar, and Thiosulfate Citrate Bile Salts Sucrose Agar). Surface samples were collected from the faucet, basin, and drain and directly spread on the media plates. Isolates were identified using standard bacteriological and biochemical tests. Kirby-Bauer disk diffusion method was performed using 21 antibiotic disks from 10 different antibiotic classes. They included ampicillin (AMP), amoxicillin (AML), piperacillin-tazobactam (TZP), cefipime (FEP), cefoxitin (FOX), ceftazidime (CAZ), ceftriaxone (CRO), imipenem (IMP), meropenem (MEM), ciprofloxacin (CIP), moxifloxacin (MXF), levofloxacin (LEV), amikacin (AK), gentamicin (CN), tigecycline (TGC), aztreonam (ATM), erythromycin (E), clindamycin (DA), rifampicin (RD), colistin (CT), and chloramphenicol (C). The results were interpreted according to EUCAST guidelines for the antibiogram of the isolates; 38 isolates were selected out of 162 based on different parameters for genotyping and detection of six beta-lactamase genes (blaSHV, blaTEM, blaCTX-M, blaOXA, blaKPC, blaNDM). Results Among these 162 isolates, 82 were obtained from water sources and 80 were collected from surfaces (faucet, basin, drain). The isolates included a variety of bacteria including Aeromonas spp. (20%), Klebsiella spp. (13%), Staphylococcus aureus (13%), Pseudomonas spp.(10%), Escherichia coli (9%), Vibrio spp. (8%), Enterococcus spp. (6%), Shigella spp. (6%), Salmonella spp. (4%), Acinetobacter spp. (3%), Staphylococcus epidermitis (3%), Streptococci spp. (2%), Proteus spp. (1%), Citrobacter spp. (1%), and Serratia spp. (1%). A diverse range of microbes were identified including clinically relevant bacteria, which shows that the urban water cycle is already contaminated with multidrug-resistant microflora of the hospital settings. Macrolide and lincosamide showed the highest resistance followed by penicillin, monobactam, and cephalosporins. blaSHV and blaTEM were prevalent in samples. blaNDM was also found which manifests as a real threat since it causes resistance against carbapenems and colistin, antibiotics reserved as a last resort against infections. Conclusions This study presented the ground reality of antibiotic resistance in Pakistan and how its subsequent spread poses a great threat to the strides made in the field of medicine and public health. Strict regulations regarding antibiotic usage, hospital effluent, and urban water sanitation must be imposed to curb the devastating effects of this increasing phenomenon.

Genomic Investigation of Methicillin-Resistant Staphylococcus aureus ST113 Strains Isolated from Tertiary Care Hospitals in Pakistan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multi-drug resistant and opportunistic pathogen. The emergence of new clones of MRSA in both healthcare settings and the community warrants serious attention and epidemiological surveillance. However, epidemiological data of MRSA isolates from Pakistan are limited. We performed a whole-genome-based comparative analysis of two (P10 and R46) MRSA strains isolated from two provinces of Pakistan to understand the genetic diversity, sequence type (ST), and distribution of virulence and antibiotic-resistance genes. The strains belong to ST113 and harbor the SCCmec type IV encoding mecA gene. Both the strains contain two plasmids, and three and two complete prophage sequences are present in P10 and R46, respectively. The specific antibiotic resistance determinants in P10 include two aminoglycoside-resistance genes, aph(3')-IIIa and aad(6), a streptothrin-resistance gene sat-4, a tetracycline-resistance gene tet(K), a mupirocin-resistance gene mupA, a point mutation in fusA conferring resistance to fusidic acid, and in strain R46 a specific plasmid associated gene ant(4')-Ib. The strains harbor many virulence factors common to MRSA. However, no Panton-Valentine leucocidin (lukF-PV/lukS-PV) or toxic shock syndrome toxin (tsst) genes were detected in any of the genomes. The phylogenetic relationship of P10 and R46 with other prevailing MRSA strains suggests that ST113 strains are closely related to ST8 strains and ST113 strains are a single-locus variant of ST8. These findings provide important information concerning the emerging MRSA clone ST113 in Pakistan and the sequenced strains can be used as reference strains for the comparative genomic analysis of other MRSA strains in Pakistan and ST113 strains globally.

Quest for Novel Preventive and Therapeutic Options Against Multidrug-Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a critical healthcare challenge due to its ability to cause persistent infections and the acquisition of antibiotic resistance mechanisms. Lack of preventive vaccines and rampant drug resistance phenomenon has rendered patients vulnerable. As new antimicrobials are in the preclinical stages of development, mining for the unexploited drug targets is also crucial. In the present study, we designed a B- and T-cell multi-epitope vaccine against P. aeruginosa using a subtractive proteomics and immunoinformatics approach. A total of five proteins were shortlisted based on essentiality, extracellular localization, virulence, antigenicity, pathway association, hydrophilicity, and low molecular weight. These include two outer membrane porins; OprF (P13794) and OprD (P32722), a protein activator precursor pra (G3XDA9), a probable outer membrane protein precursor PA1288 (Q9I456), and a conserved hypothetical protein PA4874 (Q9HUT9). These shortlisted proteins were further analyzed to identify immunogenic and antigenic B- and T-cell epitopes. The best scoring epitopes were then further subjected to the construction of a polypeptide multi-epitope vaccine and joined with cholera toxin B subunit adjuvant. The final chimeric construct was docked with TLR4 and confirmed by normal mode simulation studies. The designed B- and T-cell multi-epitope vaccine candidate is predicted immunogenic in nature and has shown strong interactions with TLR-4. Immune simulation predicted high-level production of B- and T-cell population and maximal expression was ensured in E. coli strain K12. The identified drug targets qualifying the screening criteria were: UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB (G3XD23), aspartate semialdehyde dehydrogenase (Q51344), 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (Q9HV71), 3-deoxy-D-manno-octulosonic-acid transferase (Q9HUH7), glycyl-tRNA synthetase alpha chain (Q9I7B7), riboflavin kinase/FAD synthase (Q9HVM3), aconitate hydratase 2 (Q9I2V5), probable glycosyltransferase WbpH (G3XD85) and UDP-3-O-[3-hydroxylauroyl] glucosamine N-acyltransferase (Q9HXY6). For druggability and pocketome analysis crystal and homology structures of these proteins were retrieved and developed. A sequence-based search was performed in different databases (ChEMBL, Drug Bank, PubChem and Pseudomonas database) for the availability of reported ligands and tested drugs for the screened targets. These predicted targets may provide a basis for the development of reliable antibacterial preventive and therapeutic options against P. aeruginosa. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-021-10255-3.

Antimicrobial Resistance Profiling of Biofilm Forming Non Typhoidal Salmonella enterica Isolates from Poultry and Its Associated Food Products from Pakistan.

Salmonellosis caused by non-typhoidal Salmonella enterica from poultry products is a major public health concern worldwide. This study aimed at estimating the pathogenicity and antimicrobial resistance in S. enterica isolates obtained from poultry birds and their food products from different areas of Pakistan. In total, 95/370 (25.67%) samples from poultry droppings, organs, eggs, and meat were positive for Salmonella. The isolates were further identified through multiplex PCR (mPCR) as Salmonella Typhimurium 14 (14.7%), Salmonella Enteritidis 12 (12.6%), and other Salmonella spp. 69 (72.6%). The phenotypic virulence properties of 95 Salmonella isolates exhibited swimming and/or swarming motility 95 (100%), DNA degrading activity 93 (97.8%), hemolytic activity 92 (96.8%), lipase activity 87 (91.6%), and protease activity 86 (90.5%). The sopE virulence gene known for conferring zoonotic potential was detected in S. Typhimurium (92.8%), S. Enteritidis (100%), and other Salmonella spp. (69.5%). The isolates were further tested against 23 antibiotics (from 10 different antimicrobial groups) and were found resistant against fifteen to twenty-one antibiotics. All isolates showed multiple drug resistance and were found to exhibit a high multiple antibiotic-resistant (MAR) index of 0.62 to 0.91. The strong biofilm formation at 37 °C reflected their potential adherence to intestinal surfaces. There was a significant correlation between antimicrobial resistance and the biofilm formation potential of isolates. The resistance determinant genes found among the isolated strains were blaTEM-1 (59.3%), blaOxA-1 (18%), blaPSE-1 (9.5%), blaCMY-2 (43%), and ampC (8.3%). The detection of zoonotic potential MDR Salmonella in poultry and its associated food products carrying cephalosporin and quinolone resistance genes presents a major threat to the poultry industry and public health.

Antibiotic-mediated expression analysis of Shiga toxin 1 and 2 in multi-drug-resistant Shiga toxigenic Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogens, known to cause enteric infections especially diarrhea, mainly attributed to Shiga toxins (Stxs). The use of certain antibiotics for treating this infection is controversial, owing to an increased risk for producing Stxs (Stx 1 and Stx 2). Increased antibiotic resistance is also thought to be involved in the pathogenesis of STEC diseases. The purpose of this study was to analyze the effects of antibiotics on induction of Stx 1 and Stx 2 in clinical STEC isolates and to investigate the relationships between increased resistance and Stx production. Fifteen clinical isolates were treated with sub minimum inhibitory concentrations (Sub MIC) of clinically used antibiotics (ciprofloxacin, fosfomycin, tigecycline, and meropenem), and the changes in expression levels of stx1 and stx2 genes were estimated using qRT-PCR. The expressions of Shiga toxins were found to be increased up to 6.5- and eightfold under ciprofloxacin and tigecycline Sub MIC, respectively. Fosfomycin had weak induction effect of up to twofold, whereas meropenem had the weakest influence on such expression. Resistant isolates were found to be more prone to increased expression of toxins.

Immunoinformatics Driven Prediction of Multiepitopic Vaccine Against Klebsiella pneumoniae and Mycobacterium tuberculosis Coinfection and Its Validation via In Silico Expression.

Klebsiella pneumoniae and Mycobacterium tuberculosis coinfection is one of the most lethal combinations that has been becoming frequent yet, not diagnosed and reported properly. Due to the simultaneous occurrence of both infections, diagnosis is delayed leading to inadequate treatments and mortality. With the rise of MDR Klebsiella and Mycobacterium, a prophylactic and an immunotherapeutic vaccine has to be entailed for preemptive and adroit therapeutic approach. In this study, we aim to implement reverse vaccinology approach that encompasses a comprehensive evaluation of vital aspects of the pathogens to explore immunogenic epitopes against Omp A of Klebsiella and Rv1698, Rv1973 of Mtb that may help in vaccine development. The designed multi-epitopic vaccine was assessed for antigenicity, allergenicity and various physiochemical parameters. Molecular docking and simulations were executed to assess the immunogenicity and complex stability of the vaccine. The final multi-epitopic vaccine is validated to be highly immunogenic and can serve as a valuable proactive remedy for subject pathogens.

Evaluation of Pyocyanin induced systemic pathogenicity of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA) is one of the most clinically significant nosocomial infectious agents. Clinical significance of this bacterium is intensified due to the phenomenon of its natural tendency for acquiring drug resistance mechanisms. PA produces pyocyanin (PCN), an important redox-active virulence factor. PCN has been detected in higher quantities in sputum samples of PA infected Cystic Fibrosis patients. PCN producing PA strains were isolated and characterized. Genomic 16s rRNA gene segment was amplified and sequenced (GenBank accession # jx280426). PCN was extracted and purified. In silico analysis yielded permeability and cytotoxic potential of PCN in modeled cell lines. PCN has high intestinal absorption, plasma protein binding potential, and permeability across biological membranes. Oral toxicity study in in silico rodent model classified PCN in class IV 'harmful if swallowed' (ld50 0.3-2g/kg). Cytotoxicity was assessed by oxidative stress levels in different organs in balb/c mice induced by intra peritoneal PCN injection. Significant alterations in oxidative stress levels in different organs of balb/c mice were observed. Increased levels of oxidative stress were observed in lungs, and heart, lower in liver and spleen while muscle tissues showed no significant difference in comparison to control.