Development and Evaluation of an Immunoinformatics-Based Multi-Peptide Vaccine against Acinetobacter baumannii Infection.

Multi-drug-resistant (MDR) Acinetobacter baumannii is an opportunistic pathogen associated with hospital-acquired infections. Due to its environmental persistence, virulence, and limited treatment options, this organism causes both increased patient mortality and incurred healthcare costs. Thus, prophylactic vaccination could be ideal for intervention against MDR Acinetobacter infection in susceptible populations. In this study, we employed immunoinformatics to identify peptides containing both putative B- and T-cell epitopes from proteins associated with A. baumannii pathogenesis. A novel Acinetobacter Multi-Epitope Vaccine (AMEV2) was constructed using an A. baumannii thioredoxin A (TrxA) leading protein sequence followed by five identified peptide antigens. Antisera from A. baumannii infected mice demonstrated reactivity to rAMEV2, and subcutaneous immunization of mice with rAMEV2 produced high antibody titer against the construct as well as peptide components. Immunization results in increased frequency of IL-4-secreting splenocytes indicative of a Th2 response. AMEV2-immunized mice were protected against intranasal challenge with a hypervirulent strain of A. baumannii and demonstrated reduced bacterial burden at 48 h. In contrast, all mock vaccinated mice succumbed to infection within 3 days. Results presented here provide insight into the effectiveness of immunoinformatic-based vaccine design and its potential as an effective strategy to combat the rise of MDR pathogens.

Evaluation of Antibiofilm and Antiquorum Sensing Activities of Fucoidan Characterized from Padina boryana against Nosocomial Pathogens.

Modern functional chemicals that can be employed in biotechnology, pharmaceutics, and food science are a sustainable source to be found in seaweeds. The bioactivity of the majority of these marine compounds has received scant research. Fucoidan is a highly sulfated polysaccharide with a range of bioactivities, including an antipathogenic effect. There is still much to learn about the relationship between fucoidan structure and its function in pathogen infections. By employing microwave and probe sonication to create crude fucoidan, DEAE-cellulose anion-exchange chromatography was used to further purify the substance. Purified fucoidan was structurally characterized using UV-Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and NMR analysis. The results of the structural analysis demonstrate that sulfates and hydroxyl groups are present in the isolated fucoidan. There are fucose residues, according to the NMR data. The present study investigates the bioactivity of fucoidan, a polysaccharide derived from the brown algae Padina boryana, as a potent weapon against the known nosocomial diseases Proteus vulgaris and Salmonella enterica. Fluorescence microscopy was used to show that fucoidan antibiofilm action is totally effective against Proteus vulgaris and Salmonella enterica biofilm formations as well as planktonic cell growths at dosages over 25 g/mL. Here, using in vitro investigations of the possible inactivation of molecules that are regulated by acyl-homoserine lactone (AHL) in both bacterial species, we explore the antiquarum sensing and antibiofilm capabilities of fucoidan. According to the present study, extracted fucoidan from Padina boryana can be used to generate antibacterial compounds and operate as a quorum-sensing inhibitor to combat side effects and antibiotic resistance.

Inhibition of Acinetobacter baumannii Biofilm Formation Using Different Treatments of Silica Nanoparticles.

There exists a multitude of pathogens that pose a threat to human and public healthcare, collectively referred to as ESKAPE pathogens. These pathogens are capable of producing biofilm, which proves to be quite resistant to elimination. Strains of A. baumannii, identified by the "A" in the acronym ESKAPE, exhibit significant resistance to amoxicillin in vivo due to their ability to form biofilm. This study aims to inhibit bacterial biofilm formation, evaluate novel silica nanoparticles' effectiveness in inhibiting biofilm, and compare their effectiveness. Amoxicillin was utilized as a positive control, with a concentration exceeding twice that when combined with silica NPs. Treatments included pure silica NPs, silica NPs modified with copper oxide (CuO.SiO2), sodium hydroxide (NaOH.SiO2), and phosphoric acid (H3PO4.SiO2). The characterization of NPs was conducted using scanning electron microscopy (SEM), while safety testing against normal fibroblast cells was employed by MTT assay. The microtiter plate biofilm formation assay was utilized to construct biofilm, with evaluations conducted using three broth media types: brain heart infusion (BHI) with 2% glucose and 2% sucrose, Loria broth (LB) with and without glucose and sucrose, and Dulbecco's modified eagle medium/nutrient (DMEN/M). Concentrations ranging from 1.0 mg/mL to 0.06 µg/mL were tested using a microdilution assay. Results from SEM showed that pure silica NPs were mesoporous, but in the amorphous shape of the CuO and NaOH treatments, these pores were disrupted, while H3PO4 was composed of sheets. Silica NPs were able to target Acinetobacter biofilms without harming normal cells, with viability rates ranging from 61-73%. The best biofilm formation was achieved using a BHI medium with sugar supplementation, with an absorbance value of 0.35. Biofilms treated with 5.0 mg/mL of amoxicillin as a positive control alongside 1.0 mg/mL of each of the four silica treatments in isolation, resulting in the inhibition of absorbance values of 0.04, 0.13, 0.07, 0.09, and 0.08, for SiO2, CuO.SiO2, NaOH.SiO2 and H3PO4.SiO2, respectively. When amoxicillin was combined, inhibition increased from 0.3 to 0.04; NaOH with amoxicillin resulted in the lowest minimum biofilm inhibitory concentration (MBIC), 0.25 µg/mL, compared to all treatments and amoxicillin, whereas pure silica and composite had the highest MBIC, even when combined with amoxicillin, compared to all treatments, but performed better than that of the amoxicillin alone which gave the MBIC at 625 µg/mL. The absorbance values of MBIC of each treatment showed no significant differences in relation to amoxicillin absorbance value and relation to each other. Our study showed that smaller amoxicillin doses combined with the novel silica nanoparticles may reduce toxic side effects and inhibit biofilm formation, making them viable alternatives to high-concentration dosages. Further investigation is needed to evaluate in vivo activity.

Rapid and strain-specific resistance evolution of Staphylococcus aureus against inhibitory molecules secreted by Pseudomonas aeruginosa.

IMPORTANCE: Polymicrobial infections are common. In chronic infections, the different pathogens may repeatedly interact, which could spur evolutionary dynamics with pathogens adapting to one another. Here, we explore the potential of Staphylococcus aureus to adapt to its competitor Pseudomonas aeruginosa. These two pathogens frequently co-occur, and P. aeruginosa is seen as the dominant species being able to displace S. aureus. We studied three different S. aureus strains and found that all became quickly resistant to inhibitory compounds secreted by P. aeruginosa. Our experimental evolution revealed strains-specific adaptations with three main factors contributing to resistance evolution: (i) overproduction of staphyloxanthin, a molecule protecting from oxidative stress; (ii) the formation of small colony variants also protecting from oxidative stress; and (iii) alterations of membrane transporters possibly reducing toxin uptake. Our results show that species interactions can change over time potentially favoring species co-existence, which in turn could affect disease progression and treatment options.

Taking hospital pathogen surveillance to the next level.

High-throughput bacterial genomic sequencing and subsequent analyses can produce large volumes of high-quality data rapidly. Advances in sequencing technology, with commensurate developments in bioinformatics, have increased the speed and efficiency with which it is possible to apply genomics to outbreak analysis and broader public health surveillance. This approach has been focused on targeted pathogenic taxa, such as Mycobacteria, and diseases corresponding to different modes of transmission, including food-and-water-borne diseases (FWDs) and sexually transmitted infections (STIs). In addition, major healthcare-associated pathogens such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and carbapenemase-producing Klebsiella pneumoniae are the focus of research projects and initiatives to understand transmission dynamics and temporal trends on both local and global scales. Here, we discuss current and future public health priorities relating to genome-based surveillance of major healthcare-associated pathogens. We highlight the specific challenges for the surveillance of healthcare-associated infections (HAIs), and how recent technical advances might be deployed most effectively to mitigate the increasing public health burden they cause.

Identification of Efflux Pump Mutations in Pseudomonas aeruginosa from Clinical Samples.

Efflux pumps are a specialized tool of antibiotic resistance used by Pseudomonas aeruginosa to expel antibiotics. The current study was therefore conducted to examine the expression of MexAB-OprM and MexCD-OprJ efflux pump genes. In this study, 200 samples were collected from Khyber Teaching Hospital (KTH) and Hayatabad Medical Complex (HMC) in Peshawar, Pakistan. All the isolates were biochemically identified by an Analytical Profile Index kit and at the molecular level by Polymerase Chain Reaction (PCR) utilizing specific primers for the OprL gene. A total of 26 antibiotics were tested in the current study using the guidelines of the Clinical and Laboratory Standard Institute (CLSI) and high-level resistance was shown to amoxicillin-clavulanic acid (89%) and low-level to chloramphenicol (1%) by the isolates. The antibiotic-resistant efflux pump genes MexA, MexB, OprM, MexR, MexC, MexD, OprJ, and NfxB were detected in 178 amoxicillin-clavulanic acid-resistant isolates. Mutations were detected in MexA, MexB, and OprM genes but no mutation was found in the MexR gene as analyzed by I-Mutant software. Statistical analysis determined the association of antibiotics susceptibility patterns by ANOVA: Single Factor p = 0.05. The in silico mutation impact on the protein structure stability was determined via the Dynamut server, which revealed the mutations might increase the structural stability of the mutants. The docking analysis reported that MexA wild protein showed a binding energy value of -6.1 kcal/mol with meropenem and the mexA mutant (E178K) value is -6.5 kcal/mol. The mexB wild and mutant binding energy value was -5.7 kcal/mol and -8.0 kcal/mol, respectively. Efflux pumps provide resistance against a wide range of antibiotics. Determining the molecular mechanisms of resistance in P. aeruginosa regularly will contribute to the efforts against the spread of antibiotic resistance globally.

Clinico-Microbiological Correlates of Hospital-Acquired Pneumonia: A Hospital-Based Prospective Cohort Study.

BACKGROUND AND OBJECTIVES: Hospital-acquired pneumonia (HAP) is a life-threatening hospital-acquired infection contributing to poor outcomes and mortality. Though the prevalence is comparable, the burden of comorbidities and malnutrition further worsens the scenario in developing countries. Infective agents responsible for these infections vary between regions due to the variables involved. There is a dearth of data on clinico-microbiological correlates of HAP from Northern India. With this study, we aim to explore the same and add more evidence to fill the gap. METHODOLOGY: A hospital-based cohort study was done on ICU patients of the tertiary care center in Northern India including the cohort of patients obeying a strict inclusion criterion. The clinical and microbiological correlates were estimated following an appraisal of quality of study samples. RESULTS: We found that the most common clinical feature in patients with HAP was fever (82%) followed by purulent respiratory secretions (72%), tachycardia (52%), and crepitations on auscultation (38%). Approximately 86% of cases were found to be culture-positive while others were bacteriologically sterile. Gram-negative bacilli were more commonly isolated (83% Gram-negative vs 17% Gram-positive). The most common organisms isolated were Klebsiella pneumoniae, Citrobacter freundii, Escherichia coli, Acinetobacter, and Pseudomonas aeruginosa. Staphylococcus aureus was isolated from eight specimens and all isolates were susceptible to vancomycin, linezolid, teicoplanin, and tigecycline. Seven isolates were resistant to clindamycin and all 8 were resistant to macrolides and quinolones. Five strains had methicillin resistance indicating a rising burden of 'superbugs'. The most common side involved was the right side and the right middle zone was the most common zone involved. Forty-four percent of cases had a poor outcome and succumbed to the infection. CONCLUSIONS: HAP places patients at a heightened risk of mortality and manifests a distinctive clinical-microbiological profile. It is advisable to adopt a proactive stance in averting HAI by adhering to robust prophylaxis and management protocols in alignment with regional data and hospital guidelines. Despite the study's constrained sample size, it contributes significant insights specific to the region. This underscores the necessity for further exploration through analogous studies and audits in the northern part of India. Such endeavors have the potential to tailor treatment approaches for patients, ultimately enhancing overall outcomes.

Pseudomonas aeruginosa Pangenome: Core and Accessory Genes of a Highly Resourceful Opportunistic Pathogen.

In this chapter, we leverage a novel approach to assess the seamless population structure of Pseudomonas aeruginosa, using the full repertoire of genomes sequenced to date (GenBank, April 6, 2020). In order to assess the set of core functions that represents the species as well as the differences in these core functions among the phylogroups observed in the population structure analysis, we performed pangenome analyses at the species level and at the phylogroup level. The existence of the phylogroups described in the population structure analyses was supported by their different profiles of antibiotic-resistant determinants. Finally, we utilized a presence/absence matrix of protein families from the entire species to evaluate if P. aeruginosa phylogroups can be differentiated according to their accessory genomic content. Our analysis shows that the core genome of P. aeruginosa is approximately 62% of the average gene content for the species, and it is highly enriched with pathways related to the metabolism of carbohydrates and amino acids as well as cellular processes and cell maintenance. The analysis of the accessory genome of P. aeruginosa performed in this chapter confirmed not only the existence of the three phylogroups previously described in the population structure analysis, but also of 29 genetic substructures (subgroups) within the main phylogroups. Our work illustrates the utility of populations genomics pipelines to better understand highly complex bacterial species such as P. aeruginosa.

In Silico Analyses of Extracellular Proteins of Acinetobacter baumannii as Immunogenic Candidates.

Background: Acinetobacter baumannii is an important nosocomial pathogen causing high morbidity and mortality in immunocompromised patients with prolonged hospitalization. Multidrug-resistant A. baumannii infections are on the rise worldwide. Therefore, the discovery of an effective vaccine against this bacterium seems necessary as a cost-effective and preventive strategy. Methods: In this present study, 35 genomes of A. baumannii strains were considered, and the extracellular proteins were selected, maximally having one transmembrane helix with high adhesion probability and no similarity to host proteins, as immunogenic candidates using the web tool Vaxign. Subsequently, the role of these selected proteins in bacterial pathogenesis was investigated using VICMpred. Then, the major histocompatibility complex class II, linear B-cell epitopes, and conservation of epitopes were identified using the Immune Epitope Database, BepiPred, and Epitope Conservancy Analysis, respectively. Finally, the B-cell discontinuous epitopes of each protein were predicted using ElliPro and plotted on the three-dimensional structure (3D) of the proteins. The role of the unknown proteins was predicted using the STRING database. Results: In this study, eight acceptable immunogenic candidates, including FilF, FimA, putative acid phosphatase, putative exported protein, subtilisin-like serine protease, and three uncharacterized proteins, were identified in A. baumannii. Conclusions: The results of the STRING database showed that these three uncharacterized proteins play a role in nutrition (heme utilization), peptide bond cleavage (serine peptidases), and cellular processes (MlaD protein). Extracellular proteins might play a catalyst role in the outer membrane protein-based vaccine of A. baumannii. Furthermore, this study proposed a list of potent immunogenic candidates of extracellular proteins.

Ralstonia mannitolilytica: an emerging multidrug-resistant opportunistic pathogen in a tertiary care hospital setting.

Introduction: Ralstonia mannitolilytica is a rare opportunistic pathogen capable of causing a serious infection in immunocompromised patients. Our objective was to describe all cases of R. mannitolilytica bloodstream infection identified within 2 years at our tertiary care centre, focusing on clinical characteristics, risk factors, antibiotic sensitivity patterns, management and outcomes. Case Series: We compiled a descriptive case series including 14 non-duplicate R. mannitolilytica isolates obtained from bloodstream infection samples from the microbiology laboratory of a tertiary care centre from June 2019 to June 2021. All isolates were initially identified based on their morphological properties and biochemical reactions, and then underwent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) examination for confirmation of identity. Antibiotic susceptibility testing was performed using the Kirby-Bauer disc diffusion method and Vitek 2. All 14 patients presented with symptoms of fever and/or chills, and a positive blood culture for R. mannitolilytica . After 48 h of incubation, no Ralstonia growth was reported from any of the current environmental or pharmaceutical water samples. Chemotherapy (9/14), mechanical ventilation (4/14), steroid use (2/14) and diabetes mellitus (1/14) were associated risk factors in our patients. The antibiotic sensitivity panel showed maximum resistance to aminoglycosides (64.3%) and no resistance to cefoperazone/sulbactum. Patients received treatment with cefoperazone/sulbactum and meropenem or ceftazidime. Thirteen patients recovered with antibiotic therapy and one patient succumbed to his illness. Conclusion: R. mannitolilytica can cause bloodstream infections in immunocompromised patients. It is likely to be missed or underreported due to lack of clinical awareness. MALDI-TOF MS is helpful in rapid identification. R. mannitolilytica is resistant to many routinely used antibiotics, including carbapenems.

Hidden agenda of Enterococcus faecalis lifestyle transition: planktonic to sessile state.

Enterococcus faecalis, a human gastrointestinal tract commensal, is known to cause nosocomial infections. Interestingly, the pathogen's host colonization and persistent infections are possibly linked to its lifestyle changes from planktonic to sessile state. Also, the multidrug resistance and survival fitness acquired in the sessile stage of E. faecalis has challenged treatment regimes. This situation exists because of the critical role played by several root genes and their molecular branches, which are part of quorum sensing, aggregation substance, surface adhesions, stress-related response and sex pheromones in the sessile state. It is therefore imperative to decode the hidden agenda of E. faecalis and understand the significant factors influencing biofilm formation. This would, in turn, augment the development of novel strategies to tackle E. faecalis infections.

A rare occurrence of multidrug-resistant environmental Acinetobacter baumannii strains from the soil of Mangaluru, India.

Over the last decade, Acinetobacter baumannii has emerged as one of the main causes of infections acquired in the hospital setting. Outbreaks associated with this pathogen are caused mainly due to contamination and transmission in hospital territories. However, the natural habitats of A. baumannii of clinical significance still remain unclear. In this study, we highlight the isolation and identification of multidrug-resistant environmental strains of A. baumannii from the soil of Mangaluru city. All the recovered isolates were biofilm formers, and two isolates were multidrug-resistant and showed resistance to fluoroquinolone, aminoglycosides, sulfonamide, tetracycline, and carbapenems. In addition, they exhibited protease activity, and produced phospholipase C and siderophore. To the best of our knowledge, this is the first study to isolate and identify drug-resistant strains of A. baumannii from the soil.

The Involvement of the csy1 Gene in the Antimicrobial Resistance of Acinetobacter baumannii.

Acinetobacter baumannii is an important, opportunistic nosocomial pathogen that causes a variety of nosocomial infections, and whose drug resistance rate has increased in recent years. The CRISPR-Cas system exists in several bacteria, providing adaptive immunity to foreign nucleic acid invasion. This study explores whether CRISPR-Cas is related to drug resistance. Antibiotics were used to treat strains ATCC19606 and AB43, and the expression of CRISPR-related genes was found to be changed. The Csy proteins (Csy1-4) were previously detected to promote target recognition; however, the potential function of csy1 gene is still unknown. Thus, the RecAb homologous recombination system was utilized to knock out the csy1 gene from A. baumannii AB43, which carries the Type I-Fb CRISPR-Cas system, and to observe the drug resistance changes in wild-type and csy1-deleted strains. The AB43Δcsy1 mutant strain was found to become resistant to antibiotics, while the wild-type strain was sensitive to antibiotics. Moreover, transcriptome analysis revealed that the csy1 gene regulates genes encoding CRISPR-Cas-related proteins, drug-resistant efflux pumps, membrane proteins, and oxidative phosphorylation-related proteins, inhibiting antimicrobial resistance in A. baumannii. The in vitro resistance development assay revealed that the complete CRISPR-Cas system could inhibit the development of bacterial resistance. Our findings expand our understanding of the role of CRISPR-Cas csy1 gene in A. baumannii and link the CRISPR-Cas system to the biogenesis of bacterial drug-resistant structures.

Identification of novel putative immunogenic targets and construction of a multi-epitope vaccine against multidrug-resistant Corynebacterium jeikeium using reverse vaccinology approach.

The emergence of multidrug-resistant Corynebacterium jeikeium has limited treatment options and resulted in the inability to treat C. jeikeium infections, especially in immunocompromised patients. To our knowledge, no studies have been conducted to evaluate C. jeikeium antigens for vaccine development. Given the lack of effective treatments against C. jeikeium, this study aimed to identify potential immunogenic targets against C. jeikeium as a nosocomial pathogen using a reverse vaccinology approach. To achieve this goal, we performed several immuninformatics analyses, including antigenicity, allergenicity, PSI-BLAST to the human proteome, physiochemical properties, B-cell and T-cell epitopes, molecular docking, and immunosimulation. In addition, quartile scoring and prevalence assessment were used to select the most abundant immunogenic targets in different C. jeikeium strains. Finally, protein-protein interactions were performed and the multi-epitope vaccine was developed. Five putative immunogenic targets were presented as short-listed proteins in this study, including three enzymatic proteins (WP_011273969.1, WP_041626322.1, and WP_005292204.1), one protein with DUF3235 domain (WP_011273103.1), and one hypothetical protein (WP_005293648.1). Four linear B-cell epitopes of putative immunogenic targets, including WP_011273103.1 (LNSKPTPRNAAAKPKAK), WP_011273969.1 (GEGAQGSAAPADAQATANE), WP_005292204.1 (ASVSAAQKADGIAP), and WP_041626322.1 (YSKKVAEEMGVG) were selected and inserted into the mutant TbpB C-lobe protein. This platform can effectively present multiple epitopes to the immune system. However, experimental in vitro and in vivo analysis is required to confirm the safety, immunoreactivity, and efficacy of these putative immunogenic targets.

Increasing Trend of Vancomycin-resistant Enterococci Bacteremia in a Tertiary Care Hospital of South India: A Three-year Prospective Study.

Introduction: Vancomycin-resistant enterococci (VRE) are emerging as an important multidrug-resistant pathogen causing nosocomial infections, predominantly bacteremia and urinary tract infections. VRE bacteremia has caused a significant increase in the duration of the hospital stay and mortality and had caused high public health threat due to limited treatment options. Materials and methods: Between October 2017 and September 2020, all consecutive patients with culture-proven bloodstream infection with Enterococcus species, isolated for the first time, were included in the study. A total of 427 Enterococcus species were identified, and antimicrobial susceptibility tests were performed and interpreted using Clinical and Laboratory Standard Institute guidelines. Results: Of the total 427 Enterococcus species isolated, 63 (45.6%) were VRE. Among them, 51/63 (81%) were Enterococcus faecium (E. faecium) and 5/63 (8%) were Enterococcus faecalis. There was an increased trend of VRE rate in the bloodstream infections of 6.12% (2018), 13.2% (2019), and 19.2% (2020). The majority of the VRE patients [43/63 (68%)] were admitted to the intensive care units (ICUs). Vancomycin A (VanA) is the most common phenotype isolated from 51/63(81%) patients. Conclusion: This increasing trend of VRE bacteremia is a red alert to the clinicians and the infection control practitioners, so that strict antibiotic policies and proper adherence to the infection control practices can be initiated to reduce the VRE rate. How to cite this article: Sivaradjy M, Gunalan A, Priyadarshi K, Madigubba H, Rajshekar D, Sastry AS. Increasing Trend of Vancomycin-resistant Enterococci Bacteremia in a Tertiary Care Hospital of South India: A Three-year Prospective Study. Indian J Crit Care Med 2021;25(8):881-885.

Current Status of Endolysin-Based Treatments against Gram-Negative Bacteria.

The prevalence of multidrug-resistant Gram-negative bacteria is a public health concern. Bacteriophages and bacteriophage-derived lytic enzymes have been studied in response to the emergence of multidrug-resistant bacteria. The availability of tRNAs and endolysin toxicity during recombinant protein expression is circumvented by codon optimization and lower expression levels using inducible pET-type plasmids and controlled cultivation conditions, respectively. The use of polyhistidine tags facilitates endolysin purification and alters antimicrobial activity. Outer membrane permeabilizers, such as organic acids, act synergistically with endolysins, but some endolysins permeate the outer membrane of Gram-negative bacteria per se. However, the outer membrane permeation mechanisms of endolysins remain unclear. Other strategies, such as the co-administration of endolysins with polymyxins, silver nanoparticles, and liposomes confer additional outer membrane permeation. Engineered endolysins comprising domains for outer membrane permeation is also a strategy used to overcome the current challenges on the control of multidrug-resistant Gram-negative bacteria. Metagenomics is a new strategy for screening endolysins with interesting antimicrobial properties from uncultured phage genomes. Here, we review the current state of the art on the heterologous expression of endolysin, showing the potential of bacteriophage endolysins in controlling bacterial infections.

Antimicrobial Resistance Mechanisms and Virulence of Colistin- and Carbapenem-Resistant Acinetobacter baumannii Isolated from a Teaching Hospital in Taiwan.

Acinetobacter baumannii, a Gram-negative bacterium, is an important nosocomial pathogen. Colistin-resistant A. baumannii is becoming a new concern, since colistin is one of the last-line antibiotics for infections by carbapenem-resistant A. baumannii. From 452 carbapenem-resistant isolates collected in a teaching hospital in Taipei, Taiwan, we identified seven that were resistant to colistin. Carbapenem resistance in these isolates is attributed to the presence of carbapenemase gene blaOXA-23 in their genomes. Colistin resistance is presumably conferred by mutations in the sensor kinase domain of PmrB found in these isolates, which are known to result in modification of colistin target lipid A via the PmrB-PmrA-PmrC signal transduction pathway. Overexpression of pmrC, eptA, and naxD was observed in all seven isolates. Colistin resistance mediated by pmrB mutations has never been reported in Taiwan. One of the seven isolates contained three mutations in lpxD and exhibited an altered lipopolysaccharide profile, which may contribute to its colistin resistance. No significant difference in growth rates was observed between the isolates and the reference strain, suggesting no fitness cost of colistin resistance. Biofilm formation abilities of the isolates were lower than that of the reference. Interestingly, one of the isolates was heteroresistant to colistin. Four of the isolates were significantly more virulent to wax moth larvae than the reference.

Distribution of Antimicrobial Resistance and Virulence Genes within the Prophage-Associated Regions in Nosocomial Pathogens.

Prophages are often involved in host survival strategies and contribute toward increasing the genetic diversity of the host genome. Prophages also drive horizontal propagation of various genes as vehicles. However, there are few retrospective studies contributing to the propagation of antimicrobial resistance (AMR) and virulence factor (VF) genes by prophage. We extracted the complete genome sequences of seven pathogens, including ESKAPE bacteria and Escherichia coli from a public database, and examined the distribution of both the AMR and VF genes in prophage-like regions. We found that the ratios of AMR and VF genes greatly varied among the seven species. More than 70% of Enterobacter cloacae strains had VF genes, but only 1.2% of Klebsiella pneumoniae strains had VF genes from prophages. AMR and VF genes are unlikely to exist together in the same prophage region except in E. coli and Staphylococcus aureus, and the distribution patterns of prophage types containing AMR genes are distinct from those of VF gene-carrying prophage types. AMR genes in the prophage were located near transposase and/or integrase. The prophage containing class 1 integrase possessed a significantly greater number of AMR genes than did prophages with no class 1 integrase. The results of this study present a comprehensive picture of AMR and VF genes present within, or close to, prophage-like elements and different prophage patterns between AMR- or VF-encoding prophage-like elements. IMPORTANCE Although we believe phages play an important role in horizontal gene transfer in exchanging genetic material, we do not know the distribution of the antimicrobial resistance (AMR) and/or virulence factor (VF) genes in prophages. We collected different prophage elements from the complete genome sequences of seven species-Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, and Escherichia coli-and characterized the distribution of antimicrobial resistance and virulence genes located in the prophage region. While virulence genes in prophage were species specific, antimicrobial resistance genes in prophages were highly conserved in various species. An integron structure was detected within specific prophage regions such as P1-like prophage element. Maximum of 10 antimicrobial resistance genes were found in a single prophage region, suggesting that prophages act as a reservoir for antimicrobial resistance genes. The results of this study show the different characteristic structures between AMR- or VF-encoding prophages.

Diversity and Distribution of Resistance Markers in Pseudomonas aeruginosa International High-Risk Clones.

Pseudomonas aeruginosa high-risk clones are disseminated worldwide and they are common causative agents of hospital-acquired infections. In this review, we will summarize available data of high-risk P. aeruginosa clones from confirmed outbreaks and based on whole-genome sequence data. Common feature of high-risk clones is the production of beta-lactamases and among metallo-beta-lactamases NDM, VIM and IMP types are widely disseminated in different sequence types (STs), by contrast FIM type has been reported in ST235 in Italy, whereas GIM type in ST111 in Germany. In the case of ST277, it is most frequently detected in Brazil and it carries a resistome linked to blaSPM. Colistin resistance develops among P. aeruginosa clones in a lesser extent compared to other resistance mechanisms, as ST235 strains remain mainly susceptible to colistin however, some reports described mcr positive P. aeurigonsa ST235. Transferable quinolone resistance determinants are detected in P. aeruginosa high-risk clones and aac(6')-Ib-cr variant is the most frequently reported as this determinant is incorporated in integrons. Additionally, qnrVC1 was recently detected in ST773 in Hungary and in ST175 in Spain. Continuous monitoring and surveillance programs are mandatory to track high-risk clones and to analyze emergence of novel clones as well as novel resistance determinants.

Bacteriophages that infect Gram-negative bacteria as source of signal-arrest-release motif lysins.

Treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria is challenging, a potential solution for which is the use of bacteriophage-derived lytic enzymes. However, the exogenous action of bacteriophage lysins against Gram-negative bacteria is hindered due to the presence of an impermeable outer membrane in these bacteria. Nevertheless, recent research has demonstrated that some lysins are capable of permeating the outer membrane of Gram-negative bacteria with the help of signal peptides. In the present study, we investigated the genomes of 309 bacteriophages that infect Gram-negative pathogens of clinical interest in order to determine the evolutionary markers of signal peptide-containing lysins. Complete genomes displayed 265 putative lysins, of which 17 (6.41%) contained signal-arrest-release motifs and 41 (15.47%) contained cleavable signal peptides. There was no apparent relationship between host specificity and lysin diversity. Nevertheless, the evolution of lysin genes might not be independent of the rest of the bacteriophage genome once pan-genome clustering and lysin diversity appear to be correlated. In addition, signal peptide- and signal-arrest-release-containing lysins were monophyletically distributed in the protein cladogram, suggesting that the natural selection of holin-independent lysins is divergent. Our study screened 58 (21.89%) out of 265 potential candidates for in vitro experimentation against MDR bacteria.