Chemical Synthesis and Antigenic Evaluation of Inner Core Oligosaccharides from Acinetobacter baumannii Lipopolysaccharide.

Acinetobacter baumannii is currently posing a serious threat to global health. Lipopolysaccharide (LPS) is a potent virulence factor of pathogenic Gram‐negative bacteria. To explore the antigenic properties of A. baumannii LPS, four Kdo‐containing inner core glycans from A. baumannii strain ATCC 17904 were synthesized. A flexible and divergent method based on the use of the orthogonally substituted α‐Kdo‐(2→5)‐Kdo disaccharides was developed. Selective removal of different protecting groups in these key precursors and elongation of sugar chain via α‐stereocontrolled coupling with 5,7‐O‐di‐tert‐butylsilylene or 5‐O‐benzoyl protected Kdo thioglycosides and 2‐azido‐2‐deoxyglucosyl thioglycoside allowed efficient assembly of the target molecules. Glycan microarray analysis of sera from infected patients revealed that the 4,5‐branched Kdo trimer was a potential antigenic epitope, which is attractive for further immunological research to develop carbohydrate vaccines against A. baumannii.

Acinetobacter baumannii poses a serious threat to human health. Pathogenic bacterial lipopolysaccharides (LPSs) are potent immunogens for the development of antibacterial vaccines. To investigate the antigenic properties of A. baumannii LPS, five well-defined core oligosaccharide fragments from the LPS of A. baumannii SMAL and ATCC 19606 were synthesized. A divergent synthesis strategy based on orthogonally protected α-(2 → 5)-linked Kdo dimer 6 was developed. Selective exposure of different positions in this key precursor and then elongation of sugar chains via stereocontrolled formation of both 1,2-trans and 1,2-cis-2-aminoglycosidic linkages permitted the efficient synthesis of the targets. The synthetic route also highlights a 4-O and then 7-O glycosylation sequence for assembly of the novel 4,7-branched Kdo framework. Antigenicity assay using the glycan microarray technique disclosed that tetrasaccharide 3 featuring both 4,7-branch and α-(2 → 5)-Kdo-Kdo structural elements was a potential antigenic determinant.

Colistin, a cationic amphipathic polymyxin antibiotic, has been revived as a last-line therapy for Gram-negative multidrug-resistant infections. Colistin heteroresistant and -resistant Acinetobacter baumannii have prompted fears that these infections may become untreatable. The proposed ‘self-promoted uptake’ mechanism of colistin action suggests that electrostatic and hydrophobic interactions with lipopolysaccharide (LPS) facilitate permeation through the complex Gram-negative outer membrane. Proteomic and genomic characterization of colistin-resistant A. baumannii has revealed structural outer-membrane alterations, including the absence of LPS arising from mutations in lipid A biosynthesis genes (lpxA, lpxC or lpxD). To broaden our understanding of colistin action and resistance, this thesis examined the surface properties of paired colistin-susceptible and resistant A. baumannii. These properties include morphology, topography, surface charge, surface hydrophobicity, mechanical stiffness and adhesive properties, which are potentially associated with the interaction with colistin. Atomic force microscopy (AFM) studies were conducted on dried cells and live hydrated cells. Rod-shaped colistin-susceptible cells were differentiated from spherical colistin-resistant cells; the latter frequently appeared in chains or clumps, and extracellular appendages were reduced in number and length compared to susceptible cells. Cellular elongation was revealed at stationary phase for both phenotypes. Protrusions observed on colistin-susceptible cells were suggested to represent LPS bundles. The rough featureless topography of colistin-resistant cells corresponded with the loss of LPS. Surface disruption of both phenotypes illustrated in AFM images captured in air following colistin treatment (4 µg/mL) was likely accentuated by dehydration, as disruption of colistin-treated cells in liquid was not evident. High colistin concentrations (32 µg/mL) resulted in cellular aggregation, and imparted a smoothening effect to colistin-susceptible and resistant cells. Electrostatic forces mediate the initial interaction between cationic colistin and anionic lipid-A phosphoresters. Zeta potentials of bacterial cells were thus determined as a measure of surface charge. The less electronegative charge detected for colistin-resistant versus susceptible cells at mid-logarithmic phase may theoretically impede the electrostatic binding component of colistin activity. Opposing growth-phase trends were detected at stationary phase whereby colistin-susceptible cells exhibited a lower electronegative charge, while colistin-resistant cells exhibited a higher electronegative charge, in comparison to mid-logarithmic phase cells. Neutralization of the surface charge of both phenotypes by colistin and PBN (a derivative of polymyxin B (PmB) lacking the terminal N-fatty-acyl chain) occurred in a concentration-dependent manner, emphasizing the importance of the cationic polymyxin charge for antimicrobial activity. Hydrophobic interactions between colistin and LPS are proposed to mediate outer-membrane disruption. Consequently, the cell surface hydrophobicity (CSH) of A. baumannii was determined using contact angles, which describe the tendency of a water droplet to spread across a bacterial lawn on a filter. AFM images illustrated that highly porous colistin-resistant lawns were formed compared to lawns of colistin-susceptible cells. For both phenotypes, a reduction in contact angle over time paralleled a reduction in droplet volume, suggesting that results were influenced by droplet leakage through the porous bacterial lawn. Contact angles captured 0.66 sec after droplet deposition revealed significantly lower CSH for colistin-resistant versus susceptible cells at both growth phases. At stationary phase and after colistin treatment, the CSH of both phenotypes increased, which is consistent with the surface charge alterations determined by zeta potential measurements. Cellular surface modifications following colistin treatment highlighted the ability of colistin to bind to both phenotypes, creating the impetus to evaluate the interaction between polymyxins and colistin-susceptible versus -resistant A. baumannii. Investigations were also conducted using LPS from various Gram-negative strains. Structural and mechanistic deficiencies of the fluorescent dansyl-polymyxin B (DPmB) assay to determine binding affinity were identified. Polymyxin-LPS affinity was thus quantified using an improved mono-substituted fluorescent probe, [dansyl-Lys]1polymyxinB3 (DPmB3), which exhibited a comparable affinity to colistin and PmB for LPS. Thermodynamic characterization of the polymyxin-LPS interaction using isothermal titration calorimetry (ITC) revealed enthalpically driven binding of PmB and DPmB3 to LPS, attributed to electrostatic interactions. Hydrophobic association of the [dansyl-Lys]1 substituent with LPS contributed an unfavourable entropic response to the DPmB3-LPS interaction. Attempts to characterize the polymyxin interaction with colistin-susceptible versus resistant A. baumannii whole cells were unsuccessful using both the DPmB3 fluorescence assay and ITC. Finally, the force-sensing ability of the AFM was utilized to determine bacterial mechanical and adhesive properties. Measurement of bacterial spring constants indicated that colistin-susceptible cells were stiffer than LPS-deficient colistin-resistant cells at both growth phases. Multiple large adhesive peaks were noted from force curves captured on colistin-susceptible cells; these were not observed for colistin-resistant cells, which corresponds with the reduced expression of LPS and surface appendages. Colistin treatment increased cellular rigidity and caused a marked reduction in adhesion events for both phenotypes. This thesis was the first to highlight considerable alterations to the surface properties of colistin-susceptible and resistant A. baumannii as a function of growth phase and colistin treatment. These properties reflect changes to the outer-membrane structure that potentially influence the crucial binding interaction of colistin, and may contribute to the development of colistin resistance. Important insights into the mechanisms of colistin action and resistance in this problematic pathogen have been provided.

To investigate the incidence and infection regularity of ventilator-associated pneumonia (VAP) in patients undergoing tracheal intubation and to provide reference for the prevention and treatment of VAP infection in the future. A retrospective study was conducted to collect the microbial data of airway secretion cultures from 72 patients with endotracheal intubation admitted to the emergency ward of Shanghai Fifth People's Hospital from May 2020 to February 2021, and the species of microorganisms and intubation time were statistically analyzed. Among 72 patients with endotracheal intubation, males were more than females (58.33% vs. 41.67%); Patients over 60 years old accounted for 90.28%; pneumonia was the main primary disease, accounting for 58.33%. Pathogenic tests showed that: (1) 72 patients were infected with Acinetobacter baumannii (AB), Klebsiella pneumoniae (KP), and Pseudomonas aeruginosa (PA) 48 hours after intubation, 51.39% (37/72), 27.78% (20/72), and 26.39% (19/72), respectively. The infection rate of AB was significantly higher than that of KP and PA. Within 48 hours of intubation, the infection rates of AB, KP, and PA were 20.83% (15/72), 13.89% (10/72), and 4.17% (3/72), respectively. Of the 42 patients with primary pneumonia, 61.90% (26/42) were infected with one or more of the three pathogenic bacteria AB, KP, and PA 48 hours after intubation, indicating a change in the etiology of the pathogenic bacteria, with the main pathogenic bacteria transitioning from other pathogenic bacteria to AB, KP, and PA. (2) AB, KP, and PA were prone to cause late onset VAP (i.e., intubation ≥ 5 days). Respectively, among VAP patients infected with AB, late onset VAP accounted for 59.46% (22/37). Among patients infected with KP, 75.00% (15/20) had late onset VAP. Among patients infected with PA, late onset VAP accounted for 94.74% (18/19), indicating a higher proportion of late onset VAP caused by PA and KP. (3) Infection was closely related to intubation time, and the pipeline can be replaced according to the peak period of infection. AB and KP infections peaked within 4 days after intubation, reaching 57.69% (30/52) and 50.00% (15/30), respectively. It is recommended to replace the tubes or undergo sensitive antimicrobial therapy around 3-4 days after starting the machine. The proportion of PA infection after 7 days of intubation was 72.73% (16/22), and it was considered to replace the pipeline after 7 days. (4) Most of the three pathogenic bacteria, AB, KP, and PA were carbapenem resistant pathogens with multiple drug resistance. Except for PA, the infection rate of carbapenem resistant bacteria (CRAB, CRKP) was significantly higher than that of non-carbapenem resistant bacteria (AB, KP), accounting for 86.54% (45/52) and 66.67% (20/30) of the corresponding infection cases, respectively, while CRPA only accounts for 18.18% (4/22). The main differences in VAP infection caused by AB, KP, and PA pathogens are infection time, infection probability, and carbapenem resistance. Targeted prevention and treatment measures can be implemented for patients with intubation.

Objective To investigate the bacterial spectrum and drug resistance of bone infection after multiple hospitalizations. Methods A retrospective case series study was conducted on 95 patients with bone infection due to injuries admitted in the General Hospital of Shenyang Military Area from January 2009 to December 2016. There were 76 males and 34 females, with an average age of 47 years (range, 17-94 years). Bacterial culture and drug sensitivity tests were performed in 246 specimens of the infection secretions and infected tissues. The bacterial species and drug resistance data of all the specimens were statistically analyzed. The numbers and ratios of Gram-positive bacteria and Gram negative bacteria were counted according to the changes of hospitalization frequency, and the changes of drug resistance of Staphylococcus aureus after repeated hospitalizations were also recorded. Results A total of 110 pathogenic bacteria were isolated, and mixed infection was found in 19% of the bacteria. There were 61 Gram-positive bacteria (55.5%), including 35 Staphylococcus aureus [seven methicillin-resistant staphylococcus (MRSA) strains], accounting for 57% of Gram-positive strains. Other Gram positive bacteria were mainly Enterococcus faecalis and Staphylococcus epidermidis. There were 48 Gram-negative bacteria (43.6%), including 12 Pseudomonas aeruginosa strains, accounting for 25% of Gram-negative strains, nine Klebsiella pneumoniae strains, accounting for 19% of the Gram-negative strains. Staphylococcus aureus had a resistance rate to penicillin of 82%, and the major Gram positive bacteria (Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis) were all highly sensitive to vancomycin and linezolid. The major Gram-negative bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii) were highly resistant to the second generation of cephalosporins and were sensitive to carbapenem antibiotics. In 95 bone infection patients, the ratio of Gram positive bacteria to Gram negative bacteria increased from 0.98 at the first admission to 3 after repeated hospitalizations, and the ratio change was statistically significant (P<0.05). After multiple hospitalizations, the drug resistance of Staphylococcus aureus to gentamicin, ciprofloxacin, levofloxacin, and tetracycline increased gradually. The resistance rate to penicillin was even up to 100%. Conclusions The mixed infection of bone infection is common, among which Staphylococcus aureus and Staphylococcus epidermidis are the main Gram-positive pathogenic bacteria, Pseudomonas aeruginosa and Klebsiella pneumoniae are the main Gram negative pathogenic bacteria. The proportion of Gram-positive bacteria infection increased after multiple hospitalizations and became the major pathogenic bacteria. Penicillin should be avoided in the treatment of Staphylococcus aureus infection in multiple hospitalizations, and gentamicin and ciprofloxacin should be used with caution. Vancomycin or linezolid which is more sensitive is a better option. Key words: Osteomyelitis; Bacteria; Drug susceptibility test

Screening of bacterial molecules with antagonistic or adjuvant activity

Objective To investigate the expression of procalcitonin (PCT) and C-reactive protein (CRP) , and endotoxin in bloodstream infection with different microorganisms, so as to assess the value of these inflammatory cytokines in early diagnosis of sepsis in bloodstream infections patients. Methods Data of 152 septic bloodstream infected patients with 90 male and 62 female aged from 62 to 102 years and 79.2±16.3 years in average admitted from January 2012 to December 2013 were analyzed retrospectively. According to the results of blood culture, the microorganisms could be categorized into gram-negative bacteria, gram-positive bacteria and fungus groups, and the levels of serum CRP, PCT, and endotoxin were compared among these groups of bloodstream infections patients within 24 hours after admission. Results (1) A total of 152 strains of microorganisms were surveyed including 92 gram-negative strains (61.18%) , 43 gram-positive strains (28.29%) , and 16 fungal strains (10.53%) . In the gram-negative strains, Klebsiella pneumoniae (n=29) , Acinetobacter baumannii (n=24) , Escherichia coli (n=23) , Burkholderia cepacia (n=9) and Pseudomonas aeruginosa (n=4) were the most common isolates. In the Gram-positive strains, 13 strains of Staphylococcus aureus were isolated. (2) In the gram-negative bacterial bloodstream infections group, there were 60 (64.52%) patients with endotoxin positive, and there were no endotoxin positive cases with detected gram-positive bacteria and fungal bloodstream infections. The median levels of PCT were significantly different among the three groups [gram-negative strains group: 7.760 (3.365, 28.585) ng/mL, gram-positive strains group: 0.705 (0.265, 3.225) ng/mL, fungal infection group: 1.245 (0.543, 1.998) ng/mL]. In the fungal bloodstream infection group, the mean level of CRP was higher than that in other two groups [gram-negative strains group: (126.01 ± 66.53) mg/L, gram-positive strains group: (77.58±54.21) mg/L, fungal infection group: (140.14±71.21) mg/L]. (3) The receiver operating characteristic (ROC) curve of inflammatory cytokines was made for the diagnostic value in bloodstream infections. ROC curve used to distinguish between gram-positive bacterial bloodstream infections group and fungal group showed that AUCPCT+ CRP=0.791. When PCT cut-off value was 0.92 ng/mL, and CRP cut-off value was 68.00 mg/L, the sensitivity was 50% and the specificity was 95.5%. ROC curve used to distinguish between the gram-negative bacterial bloodstream infections group and fungal group showed that AUCPCT+ CRP+ LPS=0.947. When PCT cut-off value was 2.16 ng/mL and CRP cut-off value was 94.10 mg/L, and endotoxin was positive, the sensitivity was 82.8% and the specificity was 100% . ROC curve used to distinguish between gram-negative bacterial bloodstream infections group and gram-positive bacterial group showed that AUCPCT+ CRP+ LPS=0.947. When PCT cut-off value was 2.68 ng/mL, CRP cut-off value was 106.5 mg/L, endotoxin was positive, the sensitivity was 74.2% and the specificity was 97.7% . Conclusions Gram-negative bacteria were the most common microorganisms in bloodstream infections in ICU patients. Compared with single inflammatory cytokine, the serum concentrations of PCT, CRP and endotoxin used together could provide more sensitivity and specificity for the early diagnosis of bloodstream infection with different microorganisms. Key words: Bloodstream infection; Sepsis; Gram-negative bacteria; Gram-positive bacteria; Fungal; Procalcitonin; C-reactive protein; Endotoxin

Phenotypic and molecular characterization of Acinetobacter baumannii isolates causing lower respiratory infections among ICU patients

Toll-like receptor 4 (TLR4) is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS) of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here, we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections). For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation.

Tuberculosis (TB) is a chronic condition that weakens the immune system, causes structural changes in the lungs, and can lead to infections by other bacterial pathogens. Very few studies have been done to understand the magnitude of co-infection with other bacterial pathogens, so this study was conducted to understand the co-infection pattern and burden.A total of 174 microbiologically confirmed pulmonary TB patients' samples, identified by cartridge-based nucleic acid amplification test, were further tested for other bacterial pathogens by culture over a period of five months from May 2023 to September 2023. The isolates' identification and drug susceptibility were performed using the VITEK 2 system (bioMérieux, Marcy-l'Étoile, France).Of the 174 pulmonary samples tested, 19 samples grew a significant amount of other bacterial pathogens, making the prevalence 10.91% (19/174). Among the pulmonary samples tested, 54.59% were sputum, 38.5% were bronchoalveolar lavage, and 6.89% were endotracheal aspirate. Additionally, 70.11% of the patients tested were in the age group of 19-60 years. Of the patients who had co-infection, 94.73% (18/19) were male. The most common bacterial infection was caused by Pseudomonas aeruginosa, which was identified in 36.84% of the co-infection cases (7/19). This was followed by Acinetobacter baumannii in 31.57% (6/19), Klebsiella pneumoniae in 26.31% (5/19), and Stenotrophomonas maltophilia in 5.28% (1/19). Acinetobacter baumannii and Klebsiella pneumoniae showed high drug resistance, ranging from 60% to 100% against various groups of drugs tested. None of the patient samples with co-infection showed rifampicin resistance. Among all the samples with co-infection, the majority (42.10%, or 8/19) had a high load of Mycobacterium tuberculosis complex detected by CBNAAT Ultra (Cepheid, Sunnyvale, California).Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae are unusual pathogens causing infection in community patients and are known to cause illness in hospitalized patients. These organisms' resistance was also similar to the resistance shown by hospital-acquired infections. This indicates that bacterial co-infection in pulmonary TB patients will be similar to the pattern of hospital-acquired infections. The high prevalence of bacterial co-infections (10.91%) in patients with pulmonary TB poses a significant challenge as these bacterial pathogens are not susceptible to anti-tubercular drugs. Therefore, comprehensive screening for other bacterial infections in all pulmonary TB patients is crucial for effective treatment and outcomes.

Caulobacter lipid A is conditionally dispensable in the absence of fur and in the presence of anionic sphingolipids.

Objective To investigate the distribution and drug resistance of bacterial pathogens isolated from orthopedic wounds. Methods Data of bacterial strains isolated from orthopedic wounds in the Third Hospital of Hebei Medical University from January 2008 to December 2012 were retrospectively analyzed. Strains were identified by using French bioMerieux Vitek32 identification system, and the drug susceptibility was tested by Kirby-Bauer method. Chi-square test for linear trend was performed to reveal the changes of distribution and drug resistance of the strains. Results A total of 2 456 bacterial strains were isolated, with 1 652 (67.26%) gram-negative bacilli, 777 (31.64%) gram-positive cocci, 26 (1.06%) fungi, and 1 (0.04%) gram-positive bacillus. The top five pathogens were Staphylococcus aureus (666 strains, 27.12%), Pseudomonas aeruginosa (606 strains, 24.67%), Acinetobacter baumannii (355 strains, 14.45%), Escherichia coli (188 strains, 7.65%) and Enterobacter cloacae (187 strains, 7.61%). The positive rate of Acinetobacter baumannii was on the rise during 2008 and 2012 (χ2=35.266, P<0.01). The rates of pan-drug resistant strains in Acinetobacter baumannii and Pseudomonas aeruginosa were 6.20% (22/355) and 0.17% (1/606), respectively. The rates of extended-spectrum β-lactamases positive strains in Escherichia coli and Klebsiella pneumoniae were 39.89% (75/188) and 29.23% (19/65), respectively. The rates of methicillin-resistant strains in Staphylococcus aureus and coagulase-negative Staphylococcus were 40.69% (271/666) and 52.38% (22/42), respectively. The rate of vancomycin-intermediate strains in Enterococci was 3.70% (2/54). The positive rate of methicillin-resistant Staphylococcus aureus was on the rise during 2008 and 2012 (χ2=18.317, P<0.01). Staphylococcus aureus were sensitive to teicoplanin, vancomycin and linezolid; Resistance rates to rifampicin and amikacin were 11.29%-33.33%; Resistance rates to penicillins and erythromycin were 76.80%-100.00%; Resistance rates to cefazolin, cefuroxime, cefoxitin, amikacin and levofloxacin were on the rise (P<0.05); And resistance rates to sulfamethoxazole (28.11%-48.35%) were on the decline in the same period (P<0.01). Resistance rates of Pseudomonas aeruginosa to imipenem, meropenem and sulfamethoxazole were on the rise (P<0.05); Resistance rates to ciprofloxacin, levofloxacin, amikacin, gentamicin and piperacillin/tazobactam were on the decline (P<0.05); Resistance rates to cefoperazone/sulbactam were the lowest (9.15%-20.51%). Resistance rates of Acinetobacter baumannii to imipenem, meropenem, levofloxacin, piperacillin/tazobactam, sulfamethoxazole were on the rise (P<0.01); Resistance rates to cefoperazone/sulbactam were the lowest (11.86%-19.70%). Escherichia coli and Enterobacter cloacae were sensitive to imipenem and meropenem, and the resistance rates to cefoperazone/sulbactam and piperacillin/tazobactam were low (0-14.29%); Resistance rates of Escherichia coli to piperacillin, cefepime, amikacin, levofloxacin, cefoperazone/sulbactam were on the decline (P<0.05); Resistance rates of Enterobacter cloacae to cefoxitin were on the rise (P<0.01), while the resistance rates to piperacillin, ceftazidime, cefoperazone, ceftriaxone, levofloxacin were on the decline (P<0.05). Conclusion During 2008 and 2012, the predominant bacterial pathogens of orthopedic wound in patients of the Third Hospital of Hebei Medical University are Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Enterobacter cloacae, and most strains are multiple drug resistant. Key words: Hospital, orthopedic; Wound infection; Pathogen; Drug resistance

Lipopolysaccharide (LPS) is normally considered to be essential for viability in Gram-negative bacteria but can be removed in Acinetobacter baumannii Mutant cells lacking this component of the outer membrane show growth and morphological defects. Here, we report that growth rates equivalent to the wild type can be achieved simply by propagation in minimal medium. The loss of LPS requires that cells rely on phospholipids for both leaflets of the outer membrane. We show that growth rate in the absence of LPS is not limited by nutrient availability but by the rate of outer membrane biogenesis. We hypothesize that because cells grow more slowly, outer membrane synthesis ceases to be rate limiting in minimal medium.IMPORTANCE Gram-negative bacteria are defined by their asymmetric outer membrane that consists of phospholipids on the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. LPS is essential in all but a few Gram-negative species; the reason for this differential essentiality is not well understood. One species that can survive without LPS, Acinetobacter baumannii, shows characteristic growth and morphology phenotypes. We show that these phenotypes can be suppressed under conditions of slow growth and describe how LPS loss is connected to the growth defects. In addition to better defining the challenges A. baumannii cells face in the absence of LPS, we provide a new hypothesis that may explain the species-dependent conditional essentiality.

Acinetobacter baumannii is an opportunistic bacterial pathogen that is the major cause of hospital-acquired infections. It has been shown that A. baumannii with high biofilm formation increases the risk of acquiring infection. In this study, the prevalence of virulence genes involved in biofilm formation was determined in 225 A. baumannii clinical isolates from three hospitals in Thailand. Most of the isolates were multidrug-resistant A. baumannii strains (86.2%). Among all isolates, 76.9% (173/225) showed biofilm formation ability. The association between biofilm forming ability and gentamicin resistance was found. The presence of virulence genes, epsA, bap, ompA, bfmS and blaPER-1 genes, was investigated by PCR. The prevalence of ompA, bfmS, bap, blaPER-1 and epsA genes among the isolated strains was 84.4%, 84%, 48%, 30.2%, respectively. Biofilm formation related genes, ompA and bap were associated with multidrug-resistant A. baumannii strains. The result of this study revealed that a high prevalence of biofilm-forming phenotypes among A. baumannii strains obtained from different hospitals. Effective strategies to prevent infection due to A. baumannii that produce biofilms are therefore needed. [Int Microbiol 19(2):121-129 (2016)].

ABSTRACTAntibiotic resistance in Acinetobacter baumannii is on the rise around the world, highlighting the urgent need for novel antimicrobial drugs. Antimicrobial peptides (AMPs) contribute to effective protection against infections by pathogens, making them the most promising options for next-generation antibiotics. Here, we report two designed, cationic, antimicrobial-derived peptides: Mt6, and its dextroisomer D-Mt6, belonging to the analogs of MAF-1, which is isolated from the instar larvae of houseflies. Both Mt6 and D-Mt6 have a broad-spectrum antimicrobial activity that is accompanied by strong antibacterial activities, especially against A. baumannii planktonic bacteria and biofilms. Additionally, the effect of D-Mt6 against A. baumannii is stable in a variety of physiological settings, including enzyme, salt ion, and hydrogen ion environments. Importantly, D-Mt6 cleans the bacteria on Caenorhabditis elegans without causing apparent toxicity and exhibits good activity in vivo. Both Mt6 and D-Mt6 demonstrated synergistic or additive capabilities with traditional antibiotics against A. baumannii, demonstrating their characteristics as potential complements to combination therapy. Scanning electron microscopy (SEM) and laser scanning confocal microscope (LSCM) experiments revealed that two analogs displayed rapid bactericidal activity by destroying cell membrane integrity. Furthermore, in lipopolysaccharide (LPS)-stimulated macrophage cells, these AMPs drastically decreased IL-1β and TNF-a gene expression and protein secretion, implying anti-inflammatory characteristics. This trait is likely due to its dual function of directly binding LPS and inhibiting the LPS-activated mitogen-activated protein kinase (MAPK) signaling pathways in macrophages. Our findings suggested that D-Mt6 could be further developed as a novel antimicrobial/anti-inflammatory agent and used in the treatment of A. baumannii infections.IMPORTANCE Around 700,000 people worldwide die each year from antibiotic-resistant pathogens. Acinetobacter baumannii in clinical specimens increases year by year, and it is developing a strong resistance to clinical drugs, which is resulting in A. baumannii becoming the main opportunistic pathogen. Antimicrobial peptides show great potential as new antibacterial drugs that can replace traditional antibiotics. In our study, Mt6 and D-Mt6, two new antimicrobial peptides, were designed based on a natural peptide that we first discovered in the hemlymphocytes of housefly larvae. Both Mt6 and D-Mt6 showed broad-spectrum antimicrobial activity, especially against A. baumannii, by damaging membrane integrity. Moreover, D-Mt6 showed better immunoregulatory activity against LPS induced inflammation through its LPS-neutralizing and suppression on MAPK signaling. This study suggested that D-Mt6 is a promising candidate drug as a derived peptide against A. baumannii.