Clinical Isolates Of Acinetobacter Baumannii Research Articles

Effect of Phenylalanine–Arginine Beta-Naphthylamide on the Values of Minimum Inhibitory Concentration of Quinolones and Aminoglycosides in Clinical Isolates of Acinetobacter baumannii

(1) Background: Acinetobacter baumannii has become the most important pathogen responsible for nosocomial infections in health systems. It expresses several resistance mechanisms, including the production of β-lactamases, changes in the cell membrane, and the expression of efflux pumps. (2) Methods: A. baumannii was detected by PCR amplification of the blaOXA-51-like gene. Antimicrobial susceptibility to fluoroquinolones and aminoglycosides was assessed using the broth microdilution technique according to 2018 CLSI guidelines. Efflux pump system activity was assessed by the addition of a phenylalanine–arginine beta-naphthylamide (PAβN) inhibitor. (3) Results: A total of nineteen A. baumannii clinical isolates were included in the study. In an overall analysis, in the presence of PAβN, amikacin susceptibility rates changed from 84.2% to 100%; regarding tobramycin, they changed from 68.4% to 84.2%; for nalidixic acid, they changed from 73.7% to 79.0%; as per ciprofloxacin, they changed from 68.4% to 73.7%; and, for levofloxacin, they stayed as 79.0% in both groups. (4) Conclusions: The addition of PAβN demonstrated a decrease in the rates of resistance to antimicrobials from the family of quinolones and aminoglycosides. Efflux pumps play an important role in the emergence of multidrug-resistant A. baumannii strains, and their inhibition may be useful as adjunctive therapy against this pathogen.

Antibacterial activity of silver doped hydroxyapatite toward multidrug-resistant clinical isolates of Acinetobacter baumannii

Bacteria Acinetobacter baumannii is a persistent issue in hospital-acquired infections due to its fast and potent development of multi-drug resistance. To address this urgent challenge, a novel biomaterial using silver (Ag+) ions within the hydroxyapatite (HAp) lattice has been developed to prevent infections in orthopedic surgery and bone regeneration applications without relying on antibiotics. The aim of the study was to examine the antibacterial activity of mono-substituted HAp with Ag+ ions and a mixture of mono-substituted HAps with Sr2+, Zn2+, Mg2+, SeO32- and Ag+ ions against the A. baumannii.The samples were prepared in the form of powder and disc and analyzed by disc diffusion, broth microdilution method, and scanning electron microscopy. The results from the disc-diffusion method have shown a strong antibacterial efficacy of the Ag-substituted and mixture of mono-substituted HAps (Sr, Zn, Se, Mg, Ag) toward several clinical isolates. The Minimal Inhibitory Concentrations for the powdered HAp samples ranged from 32 to 42 mg/L (Ag+ substituted) and 83–167 mg/L (mixture of mono-substituted), while the Minimal Bactericidal Concentrations after 24 h of contact ranged from 62.5 (Ag+) to 187.5–292 mg/L (ion mixture). The lower substitution level of Ag+ ions in a mixture of mono-substituted HAps was the cause of lower antibacterial effects measured in suspension. However, the inhibition zones and bacterial adhesion on the biomaterial surface were comparable. Overall, the clinical isolates of A. baumannii were effectively inhibited by substituted HAp samples, probably in the same amount as by other commercially available silver-doped materials, and such materials may provide a promising alternative or supplementation to antibiotic treatment in the prevention of infections associated with bone regeneration. The antibacterial activity of prepared samples toward A. baumannii was time-dependent and should be considered in potential applications.

Prevalence and Clinical Consequences of Colistin Heteroresistance and Evolution into Full Resistance in Carbapenem-Resistant Acinetobacter baumannii.

Colistin heteroresistance (HR) refers to a bacterial population comprised of several subpopulations with different levels of resistance to colistin. In this study, we discuss the classic form of HR, in which a resistant subpopulation exists within a predominantly susceptible population. We investigated the prevalence of colistin HR and its evolution into full resistance among 173 clinical carbapenem-resistant Acinetobacter baumannii isolates and examined the effect of HR on clinical outcomes. To determine HR, we performed population analysis profiling. Our results showed a high prevalence of HR (67.1%). To examine evolution of HR strains into full resistance, the HR strains were grown in colistin-containing broth, transferred onto colistin-containing plates, and colonies on these plates were transferred into colistin-free broth. Many of the HR strains (80.2%) evolved into full resistance, 17.2% reverted to HR, and 2.6% were borderline. We used logistic regression to compare 14-day clinical failure and 14-day mortality between patients infected by HR versus susceptible non-HR carbapenem-resistant A. baumannii. In the subgroup of patients with bacteremia, HR was significantly associated with 14-day mortality. IMPORTANCE To our knowledge, this is the first large-scale study to report on HR in Gram-negative bacteria. We described the prevalence of colistin HR in a large sample of carbapenem-resistant A. baumannii isolates, the evolution of many colistin HR isolates to a resistant phenotype following colistin exposure and withdrawal, and the clinical consequences of colistin HR. We found a high prevalence of HR among clinical carbapenem-resistant A. baumannii isolates; most evolved into a resistant phenotype following colistin exposure and withdrawal. In patients treated with colistin, evolution of HR A. baumannii into full resistance could lead to higher rates of treatment failure and contribute to the reservoir of colistin-resistant pathogens in health care settings.

A Rapid Method to Screen for Multi Drug Resistance (MDR) Trait in Clinical Isolates of Acinetobacter baumannii-Acyl Homoserine Lactone (AHL) Screening in CRAB and CSAB Isolates

Aims: Our proposal aimed to evaluate Acyl Homoserine Lactones (AHL) as a functional marker for Multi drug resistant (MDR) potential in clinical isolates of Acinetobacter baumannii. We investigated the AHL production potential of clinical isolates using a biosensor assay directly on a commonly used agar media.
 Place and Duration of Study: Department of Molecular Diagnostics and Biomarkers, Gleneagles Global Hospitals, Lakdikapul, Hyderabad-500004.
 Methodology: Antimicrobial drug sensitivity testing (AST) was performed on 72 clinical isolates of A. baumannii against two front-line antibiotics, Imipenem (10µg) and Meropenem (10µg), by Kirby-Bauer disk diffusion method. Production of long chain Acyl Homoserine lactone (AHLs) in the clinical isolates of A. baumannii was tested by cross streaking with the biosensor Chromobacterium violaceum mutant strain CV026 and Agrobacterium tumefaciens (NTL4pZLR4) by agar plate diffusion assay. Screening and identification of the quorum sensing mediator gene abaI was done by PCR to confirm its presence in all the 72 clinical isolates.
 Results: Out of the 72 clinical isolates, 58 were Carbapenem resistant Acinetobacter baumannii (CRAB) and 14 were Carbapenem sensitive Acinetobacter baumannii (CSAB) for AST by agar disc diffusion method. None of our isolates produced short chain AHLs whereas all the isolates could produce varying amounts of long chain AHLs. Genotypic confirmation of AHL gene was obtained by abaI gene PCR.
 Conclusion: Carbapenems are the front-line antibiotics used to treat gram negative bacterial infections in emergencies and in the critical care units of hospitals. Clinical isolates A. baumannii has innate resistance to several antibiotics due to various mechanisms, biofilms forming the first line of defense against antibiotics for the bacterium. Our study used AST to carbapenem as the leading marker for MDR, assuming the innate resistance of A. baumannii to other beta lactam antibiotics. Our study brought out certain important observations namely: a) All clinical isolates of A. baumannii produced Quorum Sensing signal molecules, the AHLs b) the clinical isolates of A. baumannii did not produce any short chain AHLs b) All the clinical isolates of A. baumannii produced long chain AHLs c) AHL production is not specific to carbapenem drug resistance because even CSAB isolates produced AHL d) AHL production is inherent to all clinical isolates of A. baumannii and it apparently indicates an underlying biofilm potential and MDR trait in these A. baumannii isolates. e) AHLs could be a universal marker for revealing MDR trait and biofilm potential in clinical microbiology AST profiling protocols.

Genomic Determinants of Pathogenicity and Antimicrobial Resistance of Nosocomial Acinetobacter baumannii Clinical Isolates of Hospitalized Patients (2019-2021) from a Sentinel Hospital in Hangzhou, China.

Acinetobacter baumannii (A. baumannii or AB) is one of the most opportunistic, nosocomial pathogens threatening public healthcare across countries. A. baumannii has become a primary growing concern due to its exceptional ability to acquire antimicrobial resistance (AMR) to multiple antimicrobial agents which is increasingly reported and more prevalent every year. Therefore, there is an urgent need to evaluate the AMR knowledge of A. baumannii for effective clinical treatment of nosocomial infections. This study aimed to investigate the clinical distribution AMR phenotypes and genotypes, and genomic characteristics of A. baumannii isolates recovered from hospitalized patients of different clinical departments of a sentinel hospital to improve clinical practices. A total of 123 clinical isolates were recovered from hospitalized patients of different clinical departments during 2019-2021 to analyze AMR patterns, and further subjected to whole-genome sequencing (WGS) investigations. Multi-locus sequence typing (MLST), as well as the presence of antimicrobial-resistant genes (ARGs), virulence factor genes (VFGs) and insertion sequences (ISs) were also investigated from WGS data. The results highlighted that A. baumannii clinical isolates had shown a high AMR rate, particularly from the intensive care unit (ICU), towards routinely used antimicrobials, ie, β-lactams and fluoroquinolones. ST2 was the most prevalent ST in the clinical isolates, it was strongly associated to the resistance of cephalosporins and carbapenems, with blaOXA-23 and blaOXA-66 being the most frequent determinants; moreover, high carrier rate of VFGs was also observed such as all strains containing the ompA, adeF, pgaC, lpsB, and bfmR genes. Acinetobacter baumannii clinical isolates are mostly ST2 with high rates of drug resistance and carrier of virulence factors. Therefore, it requires measurements to control its transmission and infection.

Detection of biofilm-related genes and antibiotic resistance in Acinetobacter baumannii isolated from clinical specimens

Abstract. Saadulla SOK, Muhammed SM. 2023. Detection of biofilm-related genes and antibiotic resistance in Acinetobacter baumannii isolated from clinical specimens. Biodiversitas 24: 1809-1816. A total of 53 Acinetobacter baumannii clinical isolates were collected from patients treated at Shar Hospital in Sulaimani city to determine antibiotic susceptibility patterns and their association between biofilm formation and the distribution of biofilm-related genes. Identification of suspected A. baumannii was carried out by conventional and molecular approaches, including 16S rRNA and blaoxa-51 gene amplification. In addition, disk diffusion and microtiter plate methods determined antibiotic susceptibility and biofilm-forming capability, respectively. Out of 53 A. baumannii clinical isolates 42 (79.24%), were Multidrug-Resistant (MDR), of which 3 (5.66%) were resistant to colistin. Overall, 33 (62.26%) of the clinical isolates were strong biofilm producers, 6 (11.32%) were moderate or weak biofilm producers, and 8 (15.09%) were non-biofilm-forming isolates. The frequency of biofilm-related genes ompA, bap, csuE, blaPER-1, and cpaA among A. baumannii strains was 92.45%, 79.24%, 73.58%, 9.43%, and 7.54%, respectively. The high frequency of multi-drug resistant strains, and their ability to form biofilms, is a potential threat to effective therapy and may increase morbidity and mortality in infected patients. Colistin appeared to be the most effective antibiotic for treating MDR A. baumannii infections.

Development mechanism of resistant population post-exposure to tigecycline in tigecycline-heteroresistant Acinetobacter baumannii strain.

Tigecycline heteroresistance is highly prevalent in Acinetobacter baumannii clinical isolates, reducing the efficacy of tigecycline treatment. This study investigated the population dynamics of A. baumannii with tigecycline heteroresistance to determine the origin of resistance that occurs over time after antibiotic exposure. Tigecycline heteroresistance was imitated by mixing tigecycline-susceptible and -resistant A. baumannii isolates in a 1:10-6 ratio, and confirmed using population analysis profiling. Growth curves and an in-vitro competition assay found no difference in bacterial fitness between tigecycline-resistant and -susceptible populations. The green fluorescent protein (GFP) expression system and flow cytometry were used to monitor the population dynamics of the heteroresistant population, while differentiating the resistant population from the susceptible population. The mimicked tigecycline heteroresistance was confirmed to be reproducible and stable without tigecycline. The GFP-expressing population (i.e. the resistant population) nearly went undetected because it only represented approximately 10-6 of the entire population. However, when the mimicked tigecycline-heteroresistant strain was treated with tigecycline, most subpopulations expressing GFP were detected. The surviving A. baumannii population, upon exposure to tigecycline, exhibited a high minimum inhibitory concentration for tigecycline, equivalent to that of tigecycline-resistant isolates that were used to mimic heteroresistance. These results indicate that the development of resistance in tigecycline-heteroresistant A. baumannii strains, resulting in decreased antibiotic efficacy, may depend on the selection of a pre-existing resistant subpopulation.

Effect of traditional Chinese medicine monomers interfering with quorum-sensing on virulence factors of extensively drug-resistant Acinetobacter baumannii.

The antimicrobial resistance of Acinetobacter baumannii (A. baumannii) clinical isolates has emerged as a great threat to public health. Quorum sensing (QS) is one of the resistance mechanisms for drug-resistant A. baumannii. Interfering with QS is a promising strategy to combat infections caused by drug-resistant bacteria. This study explored the QS inhibition ability of thirty-four traditional Chinese medicine monomers (TCMMs) and assessed the effect of QS inhibitors (QSIs) on the virulence factors of twelve extensively drug-resistant A. baumannii (XDRAB) strains. Nine traditional Chinese medicine monomers, such as caffeic acid, cinnamic acid, and myricetin, were found to be able to inhibit the bacterial QS. Then, at 1/8 of the minimal inhibitory concentration, we found that these QSIs inhibited extensively drug-resistant A. baumannii adhesion and biofilm formation and downregulated the expression levels of virulence-associated genes (abaI, abaR, csuE, pgaA, and bap). In conclusion, nine traditional Chinese medicine monomers have QS inhibitory activity and may downregulate QS genes to interfere with the QS system, which could inhibit the expression of extensively drug-resistant A. baumannii virulence factors. These results suggest that traditional Chinese medicine monomers could develop as novel anti-virulence compounds to control extensively drug-resistant A. baumannii infections.

Molecular characterisation of colistin and carbapenem-resistant clinical isolates of Acinetobacter baumannii from Southeast Europe

ObjectivesTo characterise 11 colistin- and carbapenem-resistant Acinetobacter baumannii isolates recently emerging in hospital settings. MethodsA. baumannii isolates were collected from hospitalised patients under colistin treatment in three countries of Southeast Europe: Turkey, Croatia, and Bosnia and Herzegovina. Isolates were identified using molecular methods. ResultsIsolates from Turkey and Croatia belong to the sequence types ST195 or ST281 of the clone lineage 2, while the single isolate from Bosnia and Herzegovina belongs to the ST231 of clone lineage 1. All isolates turned out to be highly resistant to colistin (MIC ≥ 16 mg/L) and have point mutations in pmrCAB operon genes. The colistin-resistant isolate from Bosnia and Herzegovina had a unique P170L point mutation in the pmrB gene and the R125H point mutation in the pmrC gene. The L20S mutation in the pmrA gene was detected only in isolates from Croatia and has never been reported before in isolates from this country. ConclusionColistin resistance in A. baumannii in hospitalised patients receiving colistin treatment is a result of chromosomal mutations. The pattern of point mutations in pmrCAB genes suggests a spread of specific colistin-resistant isolates within the hospital.

Detection of PgaABCD Proteins in Biofilm Producing Acinetobacter Baumannii Isolates

Acinetobacter baumannii is a common cause of nosocomial infections. This bacterium is able to survive in hostile environments (desiccation, antimicrobial therapies, nutrient unavailability) beside colonization biotic and abiotic surfaces and form biofilm in hospitals and long-term care institutions. Biofilm is a three-dimensional structure of a multicellular complex in which the cells are embedded in an Extracellular Polymeric Substance (EPS) that was produced by the organism itself. Biofilm matrix contains proteins, ions, nucleic acids, and polysaccharide polymers. The main factor that leads to biofilm formation in A. baumannii is the creation of the exopolysaccharide poly-1,6-N-acetylglucosamine (PNAG), which is a virulence factor required for adhesion and aggregation by many Gram-negative bacteria. Many studies indicated that PNAG is an important factor to keep the integrity of A. baumannii biofilms in a more dynamic and stressful environment. A cluster of four genes (pgaA, pgaB, pgaC, and pgaD) are responsible for PNAG, which is encoded by pgaABCD locus. Therefore, the aim of this work was to detect proteins encoded by this locus using SDS-PAGE gel in A. baumannii isolates. Using PCR analysis to detect 16S rRNA concluded that all 24 isolates showed a positive amplification with 242bp. Acinetobacter baumannii clinical isolates showed high resistance percentage to ampicillin-sulbactam (AMS) and ceftazidime (CAZ) with 100% and 91% respectively. Furthermore, the isolates showed 83.3% for cefepime (FEP) and 70.8% for amikacin (AK), while the isolates showed a variable resistance percentage toward other antibiotics. By using Congo red method indicated that 66.7% were positive to produce biofilm and 33.3 were non-forming biofilm. Protein sequences alignment showed 99%, 99%, 100% and 99% identity for PgaA, PgaB, PgaC and PgaD respectively with protein GenBank database.

Five copper homeostasis gene clusters encode the Cu-efflux resistome of the highly copper-tolerant Methylorubrum extorquens AM1.

In the last decade, the use of copper has reemerged as a potential strategy to limit healthcare-associated infections and to control the spread of multidrug-resistant pathogens. Numerous environmental studies have proposed that most opportunistic pathogens have acquired antimicrobial resistance in their nonclinical primary habitat. Thus, it can be presumed that copper-resistant bacteria inhabiting a primary commensal niche might potentially colonize clinical environments and negatively affect the bactericidal efficacy of Cu-based treatments. The use of copper in agricultural fields is one of the most important sources of Cu pollution that may exert selection pressure for the increase of copper resistance in soil and plant-associated bacteria. To assess the emergence of copper-resistant bacteria in natural habitats, we surveyed a laboratory collection of bacterial strains belonging to the order Rhizobiales. This study proposes that Methylorubrum extorquens AM1 is an environmental isolate well adapted to thrive in copper-rich environments that could act as a reservoir of copper resistance genes. The minimal inhibitory concentrations (MICs) of CuCl2 were used to estimate the copper tolerance of eight plant-associated facultative diazotrophs (PAFD) and five pink-pigmented facultative methylotrophs (PPFM) belonging to the order Rhizobiales presumed to come from nonclinical and nonmetal-polluted natural habitats based on their reported source of isolation. Their sequenced genomes were used to infer the occurrence and diversity of Cu-ATPases and the copper efflux resistome of Mr. extorquens AM1. These bacteria exhibited minimal inhibitory concentrations (MICs) of CuCl2 ranging between 0.020 and 1.9 mM. The presence of multiple and quite divergent Cu-ATPases per genome was a prevalent characteristic. The highest copper tolerance exhibited by Mr. extorquens AM1 (highest MIC of 1.9 mM) was similar to that found in the multimetal-resistant model bacterium Cupriavidus metallidurans CH34 and in clinical isolates of Acinetobacter baumannii. The genome-predicted copper efflux resistome of Mr. extorquens AM1 consists of five large (6.7 to 25.7 kb) Cu homeostasis gene clusters, three clusters share genes encoding Cu-ATPases, CusAB transporters, numerous CopZ chaperones, and enzymes involved in DNA transfer and persistence. The high copper tolerance and the presence of a complex Cu efflux resistome suggest the presence of relatively high copper tolerance in environmental isolates of Mr. extorquens.

Meropenem in combination with baicalein exhibits synergism against extensively drug resistant and pan-drug-resistant Acinetobacter baumannii clinical isolates in vitro.

Several studies have demonstrated that the effectiveness of carbapenems against drug-resistant Acinetobacter baumannii infections has been decreasing. Combination therapy with two or more drugs is currently under investigation to overcome the emerging resistance against carbapenems. In this study, we tested the possible synergistic interactions of a potent antibacterial flavonoid, baicalein, with meropenem to illustrate this duo's antibacterial and antibiofilm effects on 15 extensively drug resistant or pan-drug-resistant (XDR/PDR) A. baumannii clinical isolates in vitro. Isolates included in the study were identified with MALDI-TOF MS, and antibiotic resistance patterns were studied according to EUCAST protocols. Carbapenem resistance was confirmed with the modified Hodge test, and resistance genes were also analyzed with genotypical methods. Then, checkerboard and time-kill assays were performed to analyze antibacterial synergism. Additionally, a biofilm inhibition assay was performed for screening the antibiofilm activity. To provide structural and mechanistic insights into baicalein action, protein-ligand docking, and interaction profiling calculations were conducted. Our study shed light on the remarkable potential of the baicalein-meropenem combination, since either synergistic or additive antibacterial activity was observed against every XDR/PDR A. baumannii strain in question. Furthermore, the baicalein-meropenem combination displayed significantly better antibiofilm activity in contrast to standalone use. In silico studies predicted that these positive effects arose from inhibition by baicalein of A. baumannii beta-lactamases and/or penicillin-binding proteins. Overall, our findings highlight the prospective potential benefits of baicalein in combination with meropenem for the treatment of carbapenem-resistant A. baumannii infections.

Development of a Bispecific Antibody Targeting Clinical Isolates of Acinetobacter baumannii.

We previously reported developing 2 anticapsular monoclonal antibodies (mAbs) as a novel therapy for Acinetobacter baumannii infections. We sought to determine whether a bispecific mAb (bsAb) could improve avidity and efficacy while maximizing strain coverage in one molecule. Humanized mAb 65 was cloned into a single-chain variable fragment and attached to humanized mAb C8, combining their paratopes into a single bsAb (C73). We tested bsAb C73's strain coverage, binding affinity, ex vivo opsonic activity, and in vivo efficacy compared to each mAb alone and combined. The bsAb demonstrated strain coverage, binding affinity, opsonization, and in vivo efficacy superior to either original mAb alone or combined. A humanized bsAb targeting distinct A. baumannii capsule moieties enabled potent and effective coverage of disparate A. baumannii clinical isolates. The bsAb enhances feasibility of development by minimizing the number of components of a promising novel therapeutic for these difficult-to-treat infections.

GENETIC DIVERSITY OF ACINETOBACTER BAUMANNII STRAINS ISOLATED IN DIFFERENT HOSPITALS

Objective: The genotypic analysis of the strains can provide information to evaluate the genetic relationships among strains and epidemiological investigations, and it is crucial for monitoring their circulation in different geographic regions. This study was to aiming to identify genetic similarities or dissimilarities among clinical Acinetobacter baumannii (A. baumannii) isolates from four different hospitals. Result and Discussion: In this study, 78 non-duplicate clinical isolates of A. baumannii were received from patients in the critical care units. The colistin MIC values of 24 A. baumannii strains randomly selected from four different hospitals and known to have antibiotic susceptibility were determined. These strains were genetically characterized by the Enterobacterial repetitive intergenic consensus (ERIC)-PCR method. The results of the study showed that the isolates were divided into 2 clusters (A1 and A2) and Cluster A2 was represented by a single genotype (C1) and 23 interrelated genotypes were in Cluster A1.

Screening the clinical isolates of Acinetobacter baumannii for biofilm potential/trait from a tertiary care hospital in India

Background: The multidrug resistance and biofilm potential of Acinetobacter baumannii have made it difficult for the clinicians and healthcare providers to treat and control its spread, leading to mortalities: 23% in hospitalized patients and 43% among patients in intensive care. Biofilm has become an important virulent factor for Acinetobacter baumannii as it not only protects the pathogen against the antibiotics but also enables it to evade the immune system of the host. Resistance gene transfer happens among the species and across other species in the biofilm. Knowing if the clinical isolate of A. baumannii from a patient has biofilm trait will help the clinician to make an informed, better antibiotic strategy. The relationship between biofilm trait and multidrug resistance in the clinical isolates of A. baumannii was investigated in this study. Methods: 72 clinical isolates of A. baumannii were included in this study. Antimicrobial drug sensitivity testing was performed on these isolates against two front line antibiotics: Imipenem (10µg) and Meropenem (10µg) by Kirby-Bauer disk diffusion method. Biofilm formation was monitored and estimated quantitatively in 96-well polystyrene plate. The biofilms were examined under scanning electron microscope (SEM) while some key regulatory genes of the biofilm synthetic pathway were screened by PCR to corroborate the phenotype. Results: The 72 clinical isolates were characterized for carbapenem sensitivity. They were grouped as Carbapenem Resistant (CRAB) (n=58) and Carbapenem Sensitive (CSAB) (n=14) isolates. All isolates formed biofilm in our study and based on the amount of biofilm formed they were grouped as: strong biofilm producers 83% (60/72), moderate biofilm producers 11% (08) and weak biofilm producers 6% (04). Genotyping by PCR showed certain key regulatory genes like ata, bap and csu in almost all the isolates. Morphological studies showed that the biofilms were multi-layered, closely knit and with a thick strong matrix clearly visible under SEM. Conclusions: All the isolates efficiently formed robust biofilms, 83% of which formed strong biofilms (P value < 0.05). Both CRAB and CSAB formed biofilms inferring that biofilms production is inherent trait and phenotype of these clinical isolates of A. baumannii. The genotyping confirmed the presence of certain key genes of biofilm pathway and the strong integrity of the biofilms were revealed under SEM. Our study endorses the view that biofilms constitute an inherent trait which apparently enables A. baumannii to acquire MDR phenotype.

Antibacterial and Anti-Biofilm Activities of Silver Nano Particles Conjugated to Chitosan Against Multi-Drug Resistant Bacteria.

Nowadays, most of the traditional and conventional antibiotics are not effective against drug resistant bacterial strains. The emergence and spread of drug resistant bacterial cells calls for new therapeutic agents and strategies to control and treat the infections caused by these bacteria. In this regard, the application of nano-technology in medicine is an interesting approach. Therefore, the aim of this study was to evaluate the antibacterial and anti-biofilm activities of Ag Np conjugated to chitosan against multidrug resistant isolates of Staphylococcus aureus and Acinetobacter baumannii. Synthesis of the Ag Np and chitosan Np were performed according to the Turkevich and Ionic gelation methods, respectively. Then conjugation of nanoparticles was carried out by standard method. FTR analysis and transmission electron microscopy were used for validation of nanoparticle conjugation. Twenty clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and 20 clinical isolates of carbapenem resistant Acinetobacter baumannii (CRAB) were obtained from the microbial bank of Imam Khomeini hospital of Tehran, Iran. MIC values of the nanoparticles alone and in conjugation were determined using the microbroth dilution method. Then, fractional inhibitory concentration of agents was concluded by standard method. Finally, anti-biofilm activities of the conjugated Ag Np-chitosan at sub-MIC concentrations was tested against bacterial isolates. Synthesis of Ag Np resulted in the formation of nanoparticles with 10 nm dimensions. MIC90 of the chitosan, Ag Np and their conjugated form were respectively 64, 16, and 8 μg/mL against CRAB isolates. Also, MIC90 of the tested agents, in the same order as mentioned above, were 32, 16, and 4 μg/mL against MRSA. Combination of the agents had additive (0.625) and synergistic (0.375) effects against CRAB and MRSA. Ability of biofilm formation was dramatically reduced by ¼ MIC concentration (2 μg/mL) against the CRAB and ½ MIC concentration (2 μg/mL) in the case of the MRSA isolates. Ag Np-chitosan conjugation, an ideal alternative for ineffective antibiotics, exhibits great antibacterial and anti-biofilm effects against CRAB and MRSA isolates.

Phenotypic Characterization and Heterogeneity among Modern Clinical Isolates of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogenic bacterium prioritized by WHO and CDC because of its increasing antibiotic resistance. Heterogeneity among strains represents the hallmark of A. baumannii bacteria. We wondered to what extent extensively used strains, so-called reference strains, reflect the dynamic nature and intrinsic heterogeneity of these bacteria. We analyzed multiple phenotypic traits of 43 nonredundant, modern, and multidrug-resistant, extensively drug-resistant, and pandrug-resistant clinical isolates and broadly used strains of A. baumannii. Comparison of these isolates at the genetic and phenotypic levels confirmed a high degree of heterogeneity. Importantly, we observed that a significant portion of modern clinical isolates strongly differs from several historically established strains in the light of colony morphology, cellular density, capsule production, natural transformability, and in vivo virulence. The significant differences between modern clinical isolates of A. baumannii and established strains could hamper the study of A. baumannii, especially concerning its virulence and resistance mechanisms. Hence, we propose a variable collection of modern clinical isolates that are characterized at the genetic and phenotypic levels, covering a wide range of the phenotypic spectrum, with six different macrocolony type groups, from avirulent to hypervirulent phenotypes, and with naturally noncapsulated to hypermucoid strains, with intermediate phenotypes as well. Strain-specific mechanistic observations remain interesting per se, and established "reference" strains have undoubtedly been shown to be very useful to study basic mechanisms of A. baumannii biology. However, any study based on a specific strain of A. baumannii should be compared to modern and clinically relevant isolates. IMPORTANCE Acinetobacter baumannii is a bacterium prioritized by the CDC and WHO because of its increasing antibiotic resistance, leading to treatment failures. The hallmark of this pathogen is the high heterogeneity observed among isolates, due to a very dynamic genome. In this context, we tested if a subset of broadly used isolates, considered "reference" strains, was reflecting the genetic and phenotypic diversity found among currently circulating clinical isolates. We observed that the so-called reference strains do not cover the whole diversity of the modern clinical isolates. While formerly established strains successfully generated a strong base of knowledge in the A. baumannii field and beyond, our study shows that a rational choice of strain, related to a specific biological question, should be taken into consideration. Any data obtained with historically established strains should also be compared to modern and clinically relevant isolates, especially concerning drug screening, resistance, and virulence contexts.

Detection of the blaVIM-2 Gene in Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates in Sudan

Background: Acinetobacter baumannii is a pleomorphic aerobic Gram-negative bacillus that is notorious for having multidrug resistance traits. Carbapenem-resistant Acinetobacter baumannii (CRAB) is a global concern due to its ability to retain and disseminate resistance genes, which poses a threat to the spread of resistance among bacterial communities in hospital settings. The aim of our study was to evaluate the existence of the blaVIM-2 gene in carbapenem-resistant Acinetobacter baumannii clinical isolates from Sudanese hospitals. Methods and Results: Forty clinical isolates of Acinetobacter baumannii were collected from June 2021 to April 2022. All isolates of Acinetobacter baumannii were identified via BioMérieux's Vitek-2 automated system (Marcy l'Étoile, France) and evaluated for phenotypic resistance to carbapenem, using imipenem and meropenem. The enzymatic mode of resistance was assessed by the Modified Hodge test (MHT). A real-time PCR was used to detect the presence of the blaVIM-2 gene. Of 40 isolates, 32(80%) were resistant to imipenem, 4(10%) were moderately resistant, and 4(10%) were susceptible to imipenem; 24(60%) were resistant to meropenem, 10(25%) were moderately resistant, and 6(15%) were susceptible to meropenem. MHT was 70% positive with imipenem use and 55% positive with meropenem use. Real-time PCR revealed that only 30% of the samples were positive for the blaVIM-2 gene. All blaVIM-2-positive isolates were resistant to both imipenem and meropenem. Conclusion: Resistance to carbapenem poses a serious threat, denying patients treatment options. It is essential to make continuous surveillance of these strains to prevent the development of resistant strains.

Synergetic dual antibiotics-loaded chitosan/poly (vinyl alcohol) nanofibers with sustained antibacterial delivery for treatment of XDR bacteria-infected wounds

Resistance of bacterial pathogens to conventional antibiotics has remained a significant challenge in managing post-wound infections, especially in developing countries. Here, a nanofibrous chitosan/poly (vinyl alcohol) (CS/PVA) mat was designed for controlled delivery of three different concentrations of two antibiotics (colistin/meropenem ratio of 32/64 μg/ml (AB1), 64/128 μg/ml (AB2), and 128/256 (AB3) μg/ml) with synergistic antibacterial activity against ATCC and extensively drug-resistant (XDR) Acinetobacter baumannii clinical isolates. The scaffolds showed a uniform fibrous structure with no bead formation with a sustained release of the antibiotics for one week. The elongation at break, wettability, porosity, and average fiber diameter decreased with increased antibiotics concentrations. Young's modulus and tensile strength showed a significant increase after adding antibiotics. All the constructs showed excellent in vitro cytocompatibility for fibroblasts and biocompatibility in an animal model. The antibacterial assays confirmed the dose-dependent antibacterial activity of the CS/PVA. The scaffolds loaded with AB2 and AB3 showed biocidal properties against ATCC, while only CS/PVA/AB3 had antibacterial activity against XDR clinical isolates. This study suggests the CS/PVA/AB3 nanofibrous scaffold contained 128/256 μg/ml colistin/meropenem as an excellent antibacterial wound dressing for protection of skin wounds from XDR clinical isolates and now promises to proceed with pre-clinical investigations.

SecReT6 update: a comprehensive resource of bacterial Type VI Secretion Systems.

Type VI Secretion System (T6SS) plays significant roles in microbial activities via injecting effectors into adjacent cells or environments. T6SS increasingly gained attention due to its important influence on pathogenesis, microbial competition, etc. T6SS-associated research is explosively expanding on numerous grounds that call for an efficient resource. The SecReT6 version 3 provides comprehensive information on T6SS and the interactions between T6SS and T6SS-related proteins such as T6SS regulators and T6SS effectors. To assist T6SS researches like microbial competition and regulatory mechanisms, SecReT6 v3 developed online tools for detection and analysis of T6SS and T6SS-related proteins and estimation of T6SS-dependent killing risk. We have identified a novel T6SS regulator and T6SS-dependent killing capacity in Acinetobacter baumannii clinical isolates with the aid of SecReT6 v3. 17,212 T6SSs and plentiful T6SS-related proteins in 26,573 bacterial complete genomes were also detected, analyzed and incorporated into the database. The database is freely available at https://bioinfo-mml.sjtu.edu.cn/SecReT6/ .