Search

Twitching motility is a form of bacterial surface translocation powered by the type IV pilus (T4P). It is frequently analyzed by interstitial colony expansion between agar and the polystyrene surfaces of petri dishes. In such assays, the twitching motility of Acinetobacter nosocomialis was observed with MacConkey but not Luria-Bertani (LB) agar media. One difference between these two media is the presence of bile salts as a selective agent in MacConkey but not in LB. Here, we demonstrate that the addition of bile salts to LB allowed A. nosocomialis to display twitching. Similarly, bile salts enhanced the twitching of Acinetobacter baumannii and Pseudomonas aeruginosa in LB. These observations suggest that there is a common mechanism, whereby bile salts enhance bacterial twitching and promote interstitial colony expansion. Bile salts disrupt lipid membranes and apply envelope stress as detergents. Surprisingly, their stimulatory effect on twitching appears not to be related to a bacterial physiological response to stressors. Rather, it is due to their ability to alter the physicochemical properties of a twitching surface. We observed that while other detergents promoted twitching like bile salts, stresses applied by antibiotics, including the outer membrane-targeting polymyxin B, did not enhance twitching motility. More importantly, bacteria displayed increased twitching on hydrophilic surfaces such as those of glass and tissue culture-treated polystyrene plastics, and bile salts no longer stimulated twitching on these surfaces. Together, our results show that altering the hydrophilicity of a twitching surface significantly impacts T4P functionality., Importance: The bacterial type IV pilus (T4P) is a critical virulence factor for many medically important pathogens, some of which are prioritized by the World Health Organization for their high levels of antibiotic resistance. The T4P is known to propel bacterial twitching motility, the analysis of which provides a convenient assay for T4P functionality. Here, we show that bile salts and other detergents augment the twitching of multiple bacterial pathogens. We identified the underlying mechanism as the alteration of surface hydrophilicity by detergents. Consequently, hydrophilic surfaces like those of glass or plasma-treated polystyrene promote bacterial twitching, bypassing the requirement for detergents. The implication is that surface properties, such as those of tissues and medical implants, significantly impact the functionality of bacterial T4P as a virulence determinant. This offers valuable insights for developing countermeasures against the colonization and infection by bacterial pathogens of critical importance to human health on a global scale., Competing Interests: The authors declare no conflict of interest.

The Type VI secretion system (T6SS) is a multicomponent apparatus, present in many Gram-negative bacteria, which can inhibit bacterial prey in various ecological niches. Pseudomonas aeruginosa assembles one of its three T6SS (H1-T6SS) to respond to attacks from adjacent competing bacteria. Surprisingly, repeated assemblies of the H1-T6SS, termed dueling, were described in a monoculture in the absence of an attacker strain; however, the underlying mechanism was unknown. Here, we explored the role of H2-T6SS of P. aeruginosa in triggering H1-T6SS assembly. We show that H2-T6SS inactivation in P. aeruginosa causes a significant reduction in H1-T6SS dueling and that H2-T6SS activity directly triggers retaliation by the H1-T6SS. Intraspecific competition experiments revealed that elimination of H2-T6SS in non-immune prey cells conferred protection from H1-T6SS. Moreover, we show that the H1-T6SS response is triggered independently of the characterized lipase effectors of the H2-T6SS, as well as those of Acinetobacter baylyi and Vibrio cholerae . Our results suggest that H1-T6SS response to H2-T6SS in P. aeruginosa can impact intraspecific competition, particularly when the H1-T6SS effector-immunity pairs differ between strains, and could determine the outcome of multistrain colonization.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa harbors three different Type VI secretion systems (H1, H2, and H3-T6SS), which can translocate toxins that can inhibit bacterial competitors or inflict damage to eukaryotic host cells. Unlike the unregulated T6SS assembly in other Gram-negative bacteria, the H1-T6SS in P. aeruginosa is precisely assembled as a response to various cell damaging attacks from neighboring bacterial cells. Surprisingly, it was observed that neighboring P. aeruginosa cells repeatedly assemble their H1-T6SS toward each other. Mechanisms triggering this "dueling" behavior between sister cells were unknown. In this report, we used a combination of microscopy, genetic and intraspecific competition experiments to show that H2-T6SS initiates H1-T6SS dueling. Our study highlights the interplay between different T6SS clusters in P. aeruginosa , which may influence the outcomes of multistrain competition in various ecological settings such as biofilm formation and colonization of cystic fibrosis lungs., Competing Interests: The authors declare no conflict of interest.

Pathogenic bacteria of the Acinetobacter genus pose a severe threat to human health worldwide due to their strong adaptability, tolerance, and antibiotic resistance. Most isolates of these bacteria harbor a type VI secretion system (T6SS) that allows them to outcompete co-residing microorganisms, but whether this system is involved in acquiring nutrients from preys remains less studied. In this study, we found that Ab25, a clinical isolate of Acinetobacter nosocomialis, utilizes a T6SS to kill taxonomically diverse microorganisms, including bacteria and fungi. The T6SS of Ab25 is constitutively expressed, and among the three predicted effectors, T6e1, a member of the RHS effector family, contributes the most for its antimicrobial activity. T6e1 undergoes self-cleavage, and a short carboxyl fragment with nuclease activity is sufficient to kill target cells via T6SS injection. Interestingly, strain Ab25 encodes an orphan VgrG protein, which when overexpressed blocks the firing of its T6SS. In niches such as dry plastic surfaces, the T6SS promotes prey microorganism-dependent survival of Ab25. These results reveal that A. nosocomialis employs T6SSs that are highly diverse in their regulation and effector composition to gain a competitive advantage in environments with scarce nutrient supply and competing microbes.IMPORTANCEThe type VI secretion system (T6SS) plays an important role in bacterial adaptation to environmental challenges. Members of the Acinetobacter genus, particularly A. baumannii and A. nosocomialis, are notorious for their multidrug resistance and their ability to survive in harsh environments. In contrast to A. baumannii , whose T6SS has been well-studied, few research works have focused on A. nosocomialis . In this study, we found that an A. nosocomialis strain utilizes a contitutively active T6SS to kill diverse microorganisms, including bacteria and fungi. Although T6SS structural proteins of A. nosocomialis are similar to those of A. baumannii , the effector repertoire differs greatly. Interestingly, the T6SS of the A. nosocomialis strain codes for an ophan VgrG protein, which blocks the firing of the system when overexpressed, suggesting the existence of a new regulatory mechanism for the T6SS. Importantly, although the T6SS does not provide an advantage when the bacterium is grown in nutrient-rich medium, it allows A. nosocomialis to survive better in dry surfaces that contain co-existing bacteria. Our results suggest that killing of co-residing microorganisms may increase the effectiveness of strategies designed to reduce the fitness of Acinetobacter bacteria by targeting their T6SS., Competing Interests: The authors declare no conflict of interest.

Globally, drought stress poses a significant threat to crop productivity. Improving the drought tolerance of crops with microbial biostimulants is a sustainable strategy to meet a growing population's demands. This research aimed to elucidate microbial biostimulants' (Plant Growth Promoting Rhizobacteria) role in alleviating drought stress in oil-seed crops. In total, 15 bacterial isolates were selected for drought tolerance and screened for plant growth-promoting (PGP) attributes like phosphate solubilization and production of indole-3-acetic acid, siderophore, hydrogen cyanide, ammonia, and exopolysaccharide. This research describes two PGPR strains: Acinetobacter calcoaceticus AC06 and Bacillus amyloliquefaciens BA01. The present study demonstrated that these strains (AC06 and BA01) produced abundant osmolytes under osmotic stress, including proline (2.21 and 1.75 µg ml - 1 ), salicylic acid (18.59 and 14.21 µg ml - 1 ), trehalose (28.35 and 22.74 µg mg - 1 FW) and glycine betaine (11.35 and 7.74 mg g - 1 ) respectively. AC06 and BA01 strains were further evaluated for their multifunctional performance by inoculating in Arachis hypogaea L. (Groundnut) under mild and severe drought regimes (60 and 40% Field Capacity). Inoculation with microbial biostimulants displayed distinct osmotic-adjustment abilities of the groundnut, such as growth parameters, plant biomass, photosynthetic pigments, relative water content, proline, and soluble sugar in respective to control during drought. On the other hand, plant sensitivity indexes such as electrolyte leakage and malondialdehyde (MDA) contents were decreased as well as cooperatively conferred plant drought tolerance by induced alterations in stress indicators such as catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD). Thus, Acinetobacter sp. AC06 and Bacillus sp. BA01 can be considered as osmolyte producing microbial biostimulants to simultaneously induce osmotic tolerance and metabolic changes in groundnuts under drought stress., (© 2024. The Author(s).)

Implant infection impairs osseointegration of orthopaedic implants by inducing inflammation. Acinetobacter spp. are increasingly prevalent multi-drug resistant bacteria that can cause osteomyelitis. Acinetobacter spp. can also cause inflammation and thereby inhibit osseointegration in mice. The purpose of the present study was to investigate the role of quorum sensing in this context. Therefore, wild-type bacteria were compared with an isogenic abaI mutant defective in quorum sensing in a murine osseointegration model. The abaI quorum- sensing mutant affected significantly less osseointegration and interleukin (IL) 1β levels, without detectably altering other pro-inflammatory cytokines. Wild-type bacteria had fewer effects on IL1 receptor (IL1R)-/- mice. These results indicated that quorum sensing in Acinetobacter spp. contributed to IL1β induction and the resultant inhibition of osseointegration in mice. Moreover, targeting the Gram-negative acyl-homoserine lactone quorum sensing may be particularly effective for patients with Acinetobacter spp. infections.

Bacteriophage exclusion ('BREX') phage restriction systems are found in a wide range of bacteria. Various BREX systems encode unique combinations of proteins that usually include a site-specific methyltransferase; none appear to contain a nuclease. Here we describe the identification and characterization of a Type I BREX system from Acinetobacter and the effect of deleting each BREX ORF on growth, methylation, and restriction. We identified a previously uncharacterized gene in the BREX operon that is dispensable for methylation but involved in restriction. Biochemical and crystallographic analyses of this factor, which we term BrxR ('BREX Regulator'), demonstrate that it forms a homodimer and specifically binds a DNA target site upstream of its transcription start site. Deletion of the BrxR gene causes cell toxicity, reduces restriction, and significantly increases the expression of BrxC. In contrast, the introduction of a premature stop codon into the BrxR gene, or a point mutation blocking its DNA binding ability, has little effect on restriction, implying that the BrxR coding sequence and BrxR protein play independent functional roles. We speculate that elements within the BrxR coding sequence are involved in cis regulation of anti-phage activity, while the BrxR protein itself plays an additional regulatory role, perhaps during horizontal transfer., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Gram-negative bacteria from the genus Acinetobacter are responsible for life-threating hospital-related infections such as pneumonia, septicemia, and meningitis, especially in immunocompromised patients. Worryingly, Acinetobacter have become multi- and extensively drug resistant (MDR/XDR) over the last few decades. The complement system is the first line of defense against microbes, thus it is highly important to increase our understanding of evasion mechanisms used by Acinetobacter spp. Here, we studied clinical isolates of Acinetobacter spp. (n=50), aiming to characterize their recognition by the complement system. Most isolates tested survived 1 h incubation in 30% serum, and only 8 isolates had a lower survival rate, yet none of those isolates were fully killed. Intriguingly, four isolates survived in human whole blood containing all cell component. Their survival was, however, significantly reduced. Flow cytometry analyses revealed that most of the isolates were detected by human IgG and IgM. Interestingly, we could not detect any significant concentration of deposited C1q, despite observing C4b deposition that was abolished in C1q-deficient serum, indicating transient binding of C1q to bacteria. Moreover, several isolates were recognized by MBL, with C4b deposition abolished in MBL-deficient serum. C3b was deposited on most isolates, but this was not, however, seen with respect to C5b and formation of the membrane attack complex (MAC), indicating that many isolates could avoid complement-mediated lysis. India ink staining showed that isolates were capsulated, and capsule thickness varied significantly between isolates. Studies performed on a wild-type strain and capsule mutant strains, demonstrated that the production of a capsular polysaccharide is one mechanism that mediates resistance to complement-mediated bactericidal activity by preventing MAC deposition and lysis. Our data showed that most clinical Acinetobacter spp. isolates are highly serum resistant despite being efficiently recognized by the complement system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Magda, Bettoni, Laabei, Fairley, Russo, Riesbeck and Blom.)

Kawasaki disease (KD) is a multi-systemic vasculitis of unknown etiology that occurs mainly in children, and the disturbance of gut microbiota is generally believed to cause a hyperimmune reaction triggering KD. The aim of the study was to investigate the alterations in the fecal microbiota and assess its relationship with systemic inflammation. Totally 30 KD children were enrolled and followed up for 6 months, with another group of 30 age- and sex-matched healthy children as controls. Phylotype profiles of fecal microbial communities were analyzed using 16S rRNA gene sequencing. Serum inflammatory markers were detected by flow cytometer. We showed that KD children exhibited a significant reduction in fecal microbial diversity in the acute phase compared with the healthy controls. Enterococcus, Acinetobacter, Helicobacter, Lactococcus, Staphylococcus and Butyricimonas in acute KD children were significantly higher than the healthy children. Levels of systemic inflammation biomarkers, including IL-2, IL-4, IL-6, IL-10, TNF-α, and INF-γ, were significantly elevated in the acute KD children. Altered microbiota genera Enterococcus and Helicobacter abundances were shown to be correlated positively with IL-6, which were never previously reported in KD. This study suggested that gut microbiota alteration is closely associated with systemic inflammation, which provides a new perspective on the etiology and pathogenesis of KD.

Bacterial alkane metabolism is associated with a number of cellular stresses, including membrane stress and oxidative stress, and the limited uptake of charged ions such as sulfate. In the present study, the genes ssuD and tauD in Acinetobacter oleivorans DR1 cells, which encode an alkanesulfonate monooxygenase and a taurine dioxygenase, respectively, were found to be responsible for hexadecanesulfonate (C 16 SO 3 H) and taurine metabolism, and Cbl was experimentally identified as a potential regulator of ssuD and tauD expression. The expression of ssuD and tauD occurred under sulfate-limited conditions generated during n -hexadecane degradation. Interestingly, expression analysis and knockout experiments suggested that both genes are required to protect cells against oxidative stress, including that generated by n -hexadecane degradation and H 2 O 2 exposure. Measurable levels of intracellular hexadecanesulfonate were also produced during n -hexadecane degradation. Phylogenetic analysis suggested that ssuD and tauD are mainly present in soil-dwelling aerobes within the Betaproteobacteria and Gammaproteobacteria classes, which suggests that they function as controllers of the sulfur cycle and play a protective role against oxidative stress in sulfur-limited conditions. IMPORTANCE ssuD and tauD , which play a role in the degradation of organosulfonate, were expressed during n -hexadecane metabolism and oxidative stress conditions in A. oleivorans DR1. Our study confirmed that hexadecanesulfonate was accidentally generated during bacterial n -hexadecane degradation in sulfate-limited conditions. Removal of this by-product by SsuD and TauD must be necessary for bacterial survival under oxidative stress generated during n -hexadecane degradation., (Copyright © 2020 American Society for Microbiology.)

Acinetobacter baumannii is a nosocomial pathogen capable of causing a range of diseases, including respiratory and urinary tract infections and bacteremia. Treatment options are limited due to the increasing rates of antibiotic resistance, underscoring the importance of identifying new targets for antimicrobial development. During infection, A. baumannii must acquire nutrients for replication and survival. These nutrients include carbon- and nitrogen-rich molecules that are needed for bacterial growth. One possible nutrient source within the host is amino acids, which can be utilized for protein synthesis or energy generation. Of these, the amino acid histidine is among the most energetically expensive for bacteria to synthesize; therefore, scavenging histidine from the environment is likely advantageous. We previously identified the A. baumannii histidine utilization (Hut) system as being linked to nutrient zinc homeostasis, but whether the Hut system is important for histidine-dependent energy generation or vertebrate colonization is unknown. Here, we demonstrate that the Hut system is conserved among pathogenic Acinetobacter and regulated by the transcriptional repressor HutC. In addition, the Hut system is required for energy generation using histidine as a carbon and nitrogen source. Histidine was also detected extracellularly in the murine lung, demonstrating that it is bioavailable during infection. Finally, the ammonia-releasing enzyme HutH is required for acquiring nitrogen from histidine in vitro , and strains inactivated for hutH are severely attenuated in a murine model of pneumonia. These results suggest that bioavailable histidine in the lung promotes Acinetobacter pathogenesis and that histidine serves as a crucial nitrogen source during infection., (Copyright © 2020 American Society for Microbiology.)

A bacterial strain, designated B301 T and isolated from raw chicken meat obtained from a local market in Korea, was characterized and identified using a polyphasic taxonomic approach. Cells were gram-negative, non-motile, obligate-aerobic coccobacilli that were catalase-positive and oxidase-negative. The optimum growth conditions were 30°C, pH 7.0, and 0% NaCl in tryptic soy broth. Colonies were round, convex, smooth, and cream-colored on tryptic soy agar. Strain B301 T has a genome size of 3,102,684 bp, with 2,840 protein-coding genes and 102 RNA genes. The 16S rRNA gene analysis revealed that strain B301 T belongs to the genus Acinetobacter and shares highest sequence similarity (97.12%) with A. celticus ANC 4603 T and A. sichuanensis WCHAc060041 T . The average nucleotide identity and digital DNA-DNA hybridization values for closely related species were below the cutoff values for species delineation (95-96% and 70%, respectively). The DNA G+C content of strain B301 T was 37.0%. The major respiratory quinone was Q-9, and the cellular fatty acids were primarily summed feature 3 (C 16:1 ω6c/C 16:1 ω7c), C 16:0 , and C 18:1 ω9c. The major polar lipids were phosphatidylethanolamine, diphosphatidyl-glycerol, phosphatidylglycerol, and phosphatidyl-serine. The antimicrobial resistance profile of strain B301 T revealed the absence of antibiotic-resistance genes. Susceptibility to a wide range of antimicrobials, including imipenem, minocycline, ampicillin, and tetracycline, was also observed. The results of the phenotypic, chemotaxonomic, and phylogenetic analyses indicate that strain B301 T represents a novel species of the genus Acinetobacter, for which the name Acinetobacter pullorum sp. nov. is proposed. The type strain is B301 T (=KACC 21653 T = JCM 33942 T ).

Diverse interactions among species within bacterial colonies lead to intricate spatiotemporal dynamics, which can affect their growth and survival. Here, we describe the emergence of complex structures in a colony grown from mixtures of motile and non-motile bacterial species on a soft agar surface. Time-lapse imaging shows that non-motile bacteria 'hitchhike' on the motile bacteria as the latter migrate outward. The non-motile bacteria accumulate at the boundary of the colony and trigger an instability that leaves behind striking flower-like patterns. The mechanism of the front instability governing this pattern formation is elucidated by a mathematical model for the frictional motion of the colony interface, with friction depending on the local concentration of the non-motile species. A more elaborate two-dimensional phase-field model that explicitly accounts for the interplay between growth, mechanical stress from the motile species, and friction provided by the non-motile species, fully reproduces the observed flower-like patterns., Competing Interests: LX, YC, RC, WR, JH, LT No competing interests declared, (© 2020, Xiong et al.)

Enteroendocrine cells (EECs) are specialized sensory cells in the intestinal epithelium that sense and transduce nutrient information. Consumption of dietary fat contributes to metabolic disorders, but EEC adaptations to high fat feeding were unknown. Here, we established a new experimental system to directly investigate EEC activity in vivo using a zebrafish reporter of EEC calcium signaling. Our results reveal that high fat feeding alters EEC morphology and converts them into a nutrient insensitive state that is coupled to endoplasmic reticulum (ER) stress. We called this novel adaptation 'EEC silencing'. Gnotobiotic studies revealed that germ-free zebrafish are resistant to high fat diet induced EEC silencing. High fat feeding altered gut microbiota composition including enrichment of Acinetobacter bacteria, and we identified an Acinetobacter strain sufficient to induce EEC silencing. These results establish a new mechanism by which dietary fat and gut microbiota modulate EEC nutrient sensing and signaling., Competing Interests: LY, OM, JB, MB, RL, JR No competing interests declared, (© 2019, Ye et al.)

Introduction: Acinetobacter species are short, stout, gram-negative coccobacilli, generally considered to be a relatively low-grade pathogen. However, its resistance towards multiple classes of antibiotics through an array of resistance mechanisms including its ability to form biofilm has led to its emergence as an important pathogen in hospital settings. This study was done to determine the prevalence of biofilm former and Extended-spectrum Beta-Lactamase producer among Acinetobacter species., Methods: A descriptive cross-sectional study was done in the clinical microbiology laboratory, Kathmandu Medical College from January to June 2019. Convenient sampling method was used. Ethical approval was taken from the Institutional Review Committee, Ref no. 2812201805. Preliminary identification followed by characterization of Acinetobacter species was done. Antibiotic susceptibility test was done using the Kirby-Bauer method following Clinical and Laboratory Standards Institute guidelines. Extended-spectrum Beta-Lactamase was detected by combined disc method and Biofilm detection was done using congo red agar method. Statistical Package for Social Sciences 16.0 version statistical software package was used for statistical analysis. Point estimate at 95% Confidence Interval was calculated along with frequencyand proportion for binarydata., Results: Among 108 Acinetobacter species, 86 (79.7%) Acinetobacter calcoaceticus-A. baumannii complex was seen. Seventy-eight (72%) of the isolates were multidrug-resistant, 34 (31%) of the isolates were Extended-spectrum Beta-Lactamase producer and only 10 (9.3%) of the isolates, were biofilm producers., Conclusions: Multidrug-resistant Acinetobacter spp. with the ability to produce Extended-spectrum Beta-Lactamase is prevalent in our hospital settings. Strict compliance with infection control practices is necessary to curb its spread.

Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear because pilU mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus of Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additional in vitro and in vivo data show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems., Competing Interests: The authors have declared that no competing interests exist.

We created a combined system using duckweed and bacteria to enhance the efficiency of ammonium nitrogen (NH 4 + -N) and total nitrogen (TN) removal from aquaculture wastewater. Heterotrophic nitrifying bacterium was isolated from a sediment sample at an intensive land-based aquaculture farm. It was identified as Acinetobacter sp. strain A6 based on 16S rRNA gene sequence (accession number MF767879). The NH 4 + -N removal efficiency of the strain and duckweed in culture media and sampled aquaculture wastewater at 15°C was over 99% without any accumulation of nitrite or nitrate. This was significantly higher than strain A6 or duckweed alone. Interestingly, the presence of NO 3 - increased NH 4 + -N removal rate by 35.17%. Strain A6 and duckweed had mutual growth promoting-effects despite the presence of heavy metals and antibiotics stresses. In addition, strain A6 colonized abundantly and possibly formed biofilms in the inner leaves of duckweed, and possessed indoleacetic acid (IAA)- and siderophore-producing characteristics. The mutual growth promotion between strain A6 and duckweed may be the reason for their synergistic action of N removal.

Objectives: Gram-negative bacilli are the common causative agents for community-acquired, nosocomial and opportunistic infections. The recent upsurge of biofilm, as well as beta-lactamases producing strains, have synergistically led to the extensive dissemination of multi-drug resistant gram-negative bacilli. This study was carried out with an intention to detect the biofilm formation by gram-negative bacilli and determine their antibiogram along with the detection of extended-spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) production., Results: Among 314 isolates, Escherichia coli (38%) were the predominant isolates followed by Acinetobacter spp. (20%), Klebsiella spp. (16%), and Pseudomonas spp. (12%). Overall, 197 (62.73%) of isolates were biofilm positive. 84 (26.75%) and 51 (16.24%) were confirmed as ESBL and MBL producers respectively. The association between MBL production and biofilm formation was statistically significant (χ 2  = 10.20, P value= 0.002) whereas it was insignificant between ESBL and biofilm production (χ 2  = 0.006, P-value= 0.937). Most of the biofilm and MBL producing strains were multi-drug resistant.

This study was planned to evaluate sample wise isolation and antimicrobial resistant trends of Acinetobacter spp in different departments of a tertiary care hospital. This was a transversal descriptive study, carried out in the clinical microbiology laboratory of the Allama Iqbal Medical College/ Jinnah Hospital, Lahore, Pakistan, during the period of January 2015 to December 2016. Every clinical specimen was processed for bacterial culture and antimicrobial susceptibly testing. A total of 3590 (2015=1780, 2016=1810) clinical specimens were processed. Of the total, only 54.7% were gram-negative, among these Acinetobacter spp were isolated from 10.1% and 16.5% samples respectively in 2015-16 with an overall rate of 24.3%. The highest occurrence of Acinetobacter spp isolates was reported from Intensive care units (ICU) (54%) followed by surgical units (25%) and medical units (16%). It is noteworthy that ICU and internal medicine showed the highest resistance rates, whereas, lower resistance rate was observed for the outdoor patients (OPD). Although collistin showed 0% resistant while ceftriaxone, ciprofloxacin, gentamicin, and tigecycline showed 90%, 68%, 66%, 66% and 62% resistance against Acinetobacter spp. respectively. An alarming increase in the resistance rate of meropenem, cefoperazone/sulbactam, piperacillin/ tazobactam, ciprofloxacin, and imipenem was observed from the year 2015 to 2016. This startling resistance acquired by Acinetobacter spp. within a period of one year, represent very limited therapeutic options left for the infections caused by Acinetobacter spp. Unavailability of effective drugs and limited therapeutic options enforce the health care practitioners to prescribe expensive and broad range antibiotics, which may cause harm to the patient. Therefore, it is need of an hour to better understand the antimicrobial patterns and optimize antimicrobial prescription policies for the control of multidrug-resistant Acinetobacter spp.

It is important to characterize how medically, industrially, or environmentally important bacteria adhere to surfaces in liquid flows in order to control their cell adhesion and subsequent biofilm formation. Acinetobacter sp. Tol 5 is a remarkably sticky bacterium that autoagglutinates through the adhesive nanofiber protein AtaA, which is applicable to cell immobilization in bioprocesses. In this study, the adhesion and behavior of Tol 5 cells in laminar flows were investigated using flow cell systems. Tol 5 cells autoagglutinated through AtaA and formed cell clumps during flowing. The cell clumps rather than single cells went downward due to gravity and adhered to the bottom surface. Under appropriate shear stress, a twin vortex was caused by a separated flow generated at the rear of the pre-immobilized cell clumps and carried the small cell clumps to this location, resulting in their stacking there. The rearward immobilized cell clumps developed into a large, stable aggregate with a streamlined shape, independent of cell growth. Cell clumps hardly ever developed under weak shear stress that could not generate a twin vortex and were broken up under excessively strong shear stress. These cell behaviors including the importance of clumping are interesting features in the bacterial adhesion processes.

Oil sands-affected water from mining must eventually be incorporated into the reclaimed landscape or treated and released. However, this material contains petrogenic organic compounds, such as naphthenic acids and traces of polycyclic aromatic hydrocarbons. This has raised concerns for impacts of oil sands process-affected waters on the heath of wildlife and humans downstream of receiving environments. The objective of this study was to evaluate the temporal association of disease states in fish with water chemistry of oil sands-affected waters over more than a decade and determine the pathogens associated with disease pathologies. Yellow perch (Perca flavescens) captured from nearby lakes were stocked into two experimental ponds during 1995-1997 and 2008-2010. South Bison Pond is a drainage basin that has received unextracted oil sands-contaminated material. Demonstration Pond is a constructed pond containing mature fine tailings capped with fresh water. Two disease pathologies, fin erosion for which a suspected bacterial pathogen (Acinetobacter Iwoffi) is identified, and lymphocystis (confirmed using a real-time PCR) were associated with oil sands-affected water exposure. From 1995 to 1997 pathologies were most prevalent in the South Bison Pond; however, from 2008 to 2009, disease was more frequently observed in the Demonstration Pond. CYP1A activity was 3-16 fold higher in fish from experimental ponds as compared to reference populations and this pattern was consistent across all sampling years. Bile fluorescence displayed a gradient of exposure with experimental ponds being elevated over local perch populations. Naphthenic acids decreased in the Bison Pond from approximately 12 mg/L to <4 mg/L while naphthenic acids increased in the Demonstration Pond from 6 mg/L to 12 mg/L due to tailings densification. Temporal changes in naphthenic acid levels, CYP1A activity and bile fluorescent metabolites correlate positively with incidence of disease pathologies whereas all inorganic water quality changes (major ions, pH, metals) were not associated with disease responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tilapia farming is a promising growing sector in aquaculture. Yet, there are limited studies on microbiological risks associated to tilapia farms. The aim of the present study was to analyse the bacterial communities from solid surfaces in contact with air in a tilapia farm in order to evaluate the presence of bacteria potentially toxinogenic or pathogenic to humans or animals. Samples from a local tilapia farm (tank wall, aerator, water outlets, sink and floor) were analyzed by high throughput sequencing technology. Sequences were assigned to operational taxonomic units (OTUs). Proteobacteria was the main phylum represented in most samples (except for one). Cyanobacteria were a relevant phylum in the inner wall from the fattening tank and the wet floor by the pre-fattening tank. Bacteroidetes were the second phylum in relative abundance for samples from the larval rearing tank and the pre-fattening tank and one sample from the fattening tank. Fusobacteria showed highest relative abundances in samples from the larval rearing tank and pre-fattening tank. Other phyla (Verrucomicrobia, Actinobacteria, Firmicutes, Planktomycetes, Acidobacteria, Chloroflexi, Chlorobi, Gemmatiomonadetes or Fibrobacters) had lower relative abundances. A large fraction of the reads (ranging from 43.67% to 72.25%) were assigned to uncultured bacteria. Genus Acinetobacter (mainly A. calcoaceticus/baumanni) was the predominant OTU in the aerator of the fattening tank and also in the nearby sink on the floor. The genera Cetobacterium and Bacteroides showed highest relative abundances in the samples from the larval rearing tank and the pre-fattening tank. Genera including fish pathogens (Fusobacterium, Aeromonas) were only detected at low relative abundances. Potential human pathogens other than Acinetobacter were either not detected or had very low relative abundances (< 0.01%). The results of the study suggest that the main risk factors to be monitored in tilapia farm are putative human pathogenic Acinetobacter and potential cyanotoxin-producing cyanobacteria., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Acinetobacter spp. occur naturally in many different habitats, including food, soil, and surface waters. In clinical settings, Acinetobacter poses an increasing health problem, causing infections with limited to no antibiotic therapeutic options left. The presence of human generated multidrug resistant strains is well documented but the extent to how widely they are distributed within the Acinetobacter population is unknown. In this study, Acinetobacter spp. were isolated from water samples at 14 sites of the whole course of the river Danube. Susceptibility testing was carried out for 14 clinically relevant antibiotics from six different antibiotic classes. Isolates showing a carbapenem resistance phenotype were screened with PCR and sequencing for the underlying resistance mechanism of carbapenem resistance. From the Danube river water, 262 Acinetobacter were isolated, the most common species was Acinetobacter baumannii with 135 isolates. Carbapenem and multiresistant isolates were rare but one isolate could be found which was only susceptible to colistin. The genetic background of carbapenem resistance was mostly based on typical Acinetobacter OXA enzymes but also on VIM-2. The population of Acinetobacter ( baumannii and non -baumannii ) revealed a significant proportion of human-generated antibiotic resistance and multiresistance, but the majority of the isolates stayed susceptible to most of the tested antibiotics., Competing Interests: The authors declare no conflict of interest.

Plant growth-promoting rhizobacteria (PGPR) increase plant growth and crop productivity. The inoculation of plants with a bacterial mixture (consortium) apparently provides greater benefits to plant growth than inoculation with a single bacterial strain. In the present work, a bacterial consortium was formulated containing four compatible and desiccation-tolerant strains with potential as PGPR. The formulation had one moderately (Pseudomonas putida KT2440) and three highly desiccation-tolerant (Sphingomonas sp. OF178, Azospirillum brasilense Sp7 and Acinetobacter sp. EMM02) strains. The four bacterial strains were able to adhere to seeds and colonize the rhizosphere of plants when applied in both mono-inoculation and multi-inoculation treatments, showing that they can also coexist without antagonistic effects in association with plants. The effects of the bacterial consortium on the growth of blue maize were evaluated. Seeds inoculated with either individual bacterial strains or the bacterial consortium were subjected to two experimental conditions before sowing: normal hydration or desiccation. In general, inoculation with the bacterial consortium increased the shoot and root dry weight, plant height and plant diameter compared to the non-inoculated control or mono-inoculation treatments. The bacterial consortium formulated in this work had greater benefits for blue maize plants even when the inoculated seeds underwent desiccation stress before germination, making this formulation attractive for future field applications.

Horizontal gene transfer (HGT) plays a major role in the spread of antibiotic resistance. Of particular concern are Acinetobacter baumannii bacteria, which recently emerged as global pathogens, with nosocomial mortality rates reaching 19-54% (Centers for Disease Control and Prevention, 2013; Joly Guillou, 2005; Talbot et al., 2006). Acinetobacter gains antibiotic resistance remarkably rapidly (Antunes et al., 2014; Joly Guillou, 2005), with multi drug-resistance (MDR) rates exceeding 60% (Antunes et al., 2014; Centers for Disease Control and Prevention, 2013). Despite growing concern (Centers for Disease Control and Prevention, 2013; Talbot et al., 2006), the mechanisms underlying this extensive HGT remain poorly understood (Adams et al., 2008; Fournier et al., 2006; Imperi et al., 2011; Ramirez et al., 2010; Wilharm et al., 2013). Here, we show bacterial predation by Acinetobacter baylyi increases cross-species HGT by orders of magnitude, and we observe predator cells functionally acquiring adaptive resistance genes from adjacent prey. We then develop a population-dynamic model quantifying killing and HGT on solid surfaces. We show DNA released via cell lysis is readily available for HGT and may be partially protected from the environment, describe the effects of cell density, and evaluate potential environmental inhibitors. These findings establish a framework for understanding, quantifying, and combating HGT within the microbiome and the emergence of MDR super-bugs.

Background: Immobilization of microbial cells is an important strategy for the efficient use of whole-cell catalysts because it simplifies product separation, enables the cell concentration to be increased, stabilizes enzymatic activity, and permits repeated or continuous biocatalyst use. However, conventional immobilization methods have practical limitations, such as limited mass transfer in the inner part of a gel, gel fragility, cell leakage from the support matrix, and adverse effects on cell viability and catalytic activity. We previously showed a new method for bacterial cell immobilization using AtaA, a member of the trimeric autotransporter adhesin family found in Acinetobacter sp. Tol 5. This approach is expected to solve the drawbacks of conventional immobilization methods. However, similar to all other immobilization methods, the use of support materials increases the cost of bioprocesses and subsequent waste materials., Results: We found that the stickiness of the AtaA molecule isolated from Tol 5 cells is drastically diminished at ionic strengths lower than 10 mM and that it cannot adhere in deionized water, which also inhibits cell adhesion mediated by AtaA. Cells immobilized on well plates and polyurethane foam in a salt solution were detached in deionized water by rinsing and shaking, respectively. The detached cells regained their adhesiveness in a salt solution and could rapidly be re-immobilized. The cells expressing the ataA gene maintained their adhesiveness throughout four repeated immobilization and detachment cycles and could be repeatedly immobilized to polyurethane foam by a 10-min shake in a flask. We also demonstrated that both bacterial cells and a support used in a reaction could be reused for a different type of reaction after detachment of the initially immobilized cells from the support and a subsequent immobilization step., Conclusions: We invented a unique reversible immobilization method based on the salt-dependent adhesion of the AtaA molecule that allows us to reuse bacterial cells and supports by a simple manipulation involving a deionized water wash. This mitigates problems caused by the use of support materials and greatly helps to enhance the efficiency and productivity of microbial production processes.

Aim: Implementing a mouse model of Acinetobacter baumannii (AB) digestive colonization and studying the propensity of an intestinal reservoir of AB to be at the origin of pneumonia., Materials & Methods: After a disruption of the digestive flora by piperacillin-tazobactam, two multidrug-resistant AB strains were intranasally inoculated to two cohorts of ten mice daily. For each strain, five mice were rendered transiently neutropenic., Results & Conclusion: One strain persisted several weeks in the digestive tract, even after stopping piperacillin-tazobactam injections, leading to the hypothesis that some AB strains can authentically colonize the gut. Most of the immunocompromised mice experienced clinical signs and positive lung cultures, which were associated with positive spleen cultures, an argument in favor of bacterial translocation.

Hospital-acquired infections as a result of Acinetobacter baumannii have become problematic because of high rates of drug resistance. Although neutrophils play a critical role in early protection against bacterial infection, their interactions with A. baumannii remain largely unknown. To elucidate the interactions between A. baumannii and human neutrophils, we cocultured these cells and analyzed them by microscopy and flow cytometry. We found that A. baumannii adhered to neutrophils. We next examined neutrophil and A. baumannii infiltration into Matrigel basement membranes by an in vitro transmigration assay. Neutrophils were activated by A. baumannii, and invasion was enhanced. More interestingly, A. baumannii was transported together by infiltrating neutrophils. Furthermore, we observed by live cell imaging that A. baumannii and neutrophils moved together. In addition, A. baumannii-activated neutrophils showed increased IL-8 production. The transport of A. baumannii was suppressed by inhibiting neutrophil infiltration by blocking the effect of IL-8. A. baumannii appears to use neutrophils for transport by activating these cells via IL-8. In this study, we revealed a novel bacterial transport mechanism that A. baumannii exploits human neutrophils by adhering to and inducing IL-8 release for bacterial portage. This mechanism might be a new treatment target., (© Society for Leukocyte Biology.)

The molecular and genetic basis of Acinetobacter baumannii and Acinetobacter pittii virulence remains poorly understood, and there is still lack of knowledge in host cell response to these bacteria. In this study, we have used eleven clinical Acinetobacter strains (A. baumannii n = 5; A. pittii n = 6) to unravel bacterial adherence, invasion and cytotoxicity to human lung epithelial cells. Our results showed that adherence to epithelial cells by Acinetobacter strains is scarce and cellular invasion was not truly detected. In addition, all Acinetobacter strains failed to induce any cytotoxic effect on A549 cells., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: Acinetobacter species are associated with increasing mortality due to emerging drug-resistance. Pediatric Acinetobacter infections are largely undefined in developed countries and clinical laboratory identification methods do not reliably differentiate between members of the Acinetobacter calcoaceticus-baumannii complex, leading to improper identification. Therefore we aimed to determine risk factors for invasive Acinetobacter infections within an academic, pediatric setting as well as defining microbiologic characteristics of predominant strains., Methods: Twenty-four invasive Acinetobacter isolates were collected from 2009-2013. Comparative sequence analysis of the rpoB gene was performed coupled with phenotypic characterization of antibiotic resistance, motility, biofilm production and clinical correlation., Results: Affected patients had a median age of 3.5 years, and 71 % had a central catheter infection source. rpoB gene sequencing revealed a predominance of A. pittii (45.8 %) and A. baumannii (33.3 %) strains. There was increasing incidence of A. pittii over the study. Two fatalities occurred in the A. pittii group. Seventeen percent of isolates were multi-drug resistant. A pittii and A. baumannii strains were similar in motility, but A pittii strains had significantly more biofilm production (P value = 0.018)., Conclusions: A. pittii was the most isolated species highlighting the need for proper species identification. The isolated strains had limited acute mortality in children, but the occurrence of more multi-drug resistant strains in the future is a distinct possibility, justifying continued research and accurate species identification.

Trimeric autotransporter adhesins (TAAs), cell surface proteins of Gram-negative bacteria, mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific, high adhesiveness to abiotic material surfaces as well as to biotic surfaces. AtaA is a homotrimer of polypeptides comprising 3,630 amino acids and forms long nanofibers; therefore, it is too large and structurally complex to be produced as a recombinant protein. In this study, we isolated AtaA's passenger domain (AtaA PSD), which is translocated to the cell surface through the C-terminal transmembrane domain and exhibits biological functions, using a new method. We introduced a protease recognition site and reaped AtaA nanofibers 225 nm in length from the cell surface through proteolytic cleavage with a specific protease. Biochemical and biophysical analyses of the purified native AtaA PSD revealed that it has a stable structure under alkaline and acidic conditions. Temperatures above 80 °C, which disrupted AtaA's higher-order structure but maintained the full-length AtaA polypeptide, inactivated AtaA's nonspecific adhesiveness, suggesting that the stickiness of AtaA requires its 3D structure. This finding refutes the widespread but vague speculation that large unfolded polypeptides readily stick to various surfaces.

Metabolic cross-feeding interactions are ubiquitous in natural microbial communities. However, it remains generally unclear whether the production and exchange of metabolites incurs fitness costs to the producing cells and if so, which ecological mechanisms can facilitate a cooperative exchange of metabolites among unrelated individuals. We hypothesized that positive assortment within structured environments can maintain mutualistic cross-feeding. To test this, we engineered Acinetobacter baylyi and Escherichia coli to reciprocally exchange essential amino acids. Interspecific coculture experiments confirmed that non-cooperating types were selectively favoured in spatially unstructured (liquid culture), yet disfavoured in spatially structured environments (agar plates). Both an individual-based model and experiments with engineered genotypes indicated that a segregation of cross-feeders and non-cooperating auxotrophs stabilized cooperative cross-feeding in spatially structured environments. Chemical imaging confirmed that auxotrophs were spatially excluded from cooperative benefits. Together, these results demonstrate that cooperative cross-feeding between different bacterial species is favoured in structured environments such as bacterial biofilms, suggesting this type of interactions might be common in natural bacterial communities.

In this study, we investigated differentially expressed proteins in Acinetobacter oleivorans cells during planktonic and biofilm growth by using 2-dimensional gel electrophoresis combined with matrix-assisted laser desorption time-of-flight mass spectrometry. We focused on the role of oxidative stress resistance during biofilm formation using mutants defective in alkyl hydroperoxide reductase (AhpC) because its production in aged biofilms was enhanced compared to that in planktonic cells. Results obtained using an ahpC promoter-gfp reporter vector showed that aged biofilms expressed higher ahpC levels than planktonic cells at 48 h. However, at 24 h, ahpC expression was higher in planktonic cells than in biofilms. Deletion of ahpC led to a severe growth defect in rich media that was not observed in minimal media and promoted early biofilm formation through increased production of exopolysaccharide (EPS) and EPS gene expression. Increased endogenous H2O2 production in the ahpC mutant in rich media enhanced biofilm formation, and this enhancement was not observed in the presence of antioxidants. Exogenous addition of H2O2 promoted biofilm formation in wild type cells, which suggested that biofilm development is linked to defense against H2O2. Collectively, our data showed that EPS production caused by H2O2 stress enhances biofilm formation in A. oleivorans.

Natural transformation in bacteria facilitates the uptake and genomic integration of exogenous DNA. This allows horizontal exchange of adaptive traits not easily achieved by point mutations, and has a major role in the acquisition of adaptive traits exemplified by antibiotic resistance determinants and vaccination escape. Mechanisms of DNA uptake and genomic integration are well described for several naturally transformable bacterial species; however, the selective forces responsible for its evolution and maintenance are still controversial. In this study we evolved transformation-proficient and -deficient Acinetobacter baylyi for 175 days in serial transfer cultures where stress was included. We found that natural transformation-proficient populations adapted better to active growth and early stationary phase. This advantage was offset by the reduced performance in the late stationary/death phase. We demonstrate fitness trade-offs between adaptation to active growth and survival in stationary/death phase caused by antagonistic pleiotropy. The presented data suggest that the widely held assumption that recombination speeds up adaptation by rapid accumulation of multiple adaptive mutations in the same genetic background is not sufficient to fully account for the maintenance of natural transformation in bacteria.

Confined spatial patterns of microbial distribution are prevalent in nature, such as in microbial mats, soil communities, and water stream biofilms. The symbiotic two-species consortium of Pseudomonas putida and Acinetobacter sp. strain C6, originally isolated from a creosote-polluted aquifer, has evolved a distinct spatial organization in the laboratory that is characterized by an increased fitness and productivity. In this consortium, P. putida is reliant on microcolonies formed by Acinetobacter sp. C6, to which it attaches. Here we describe the processes that lead to the microcolony pattern by Acinetobacter sp. C6. Ecological spatial pattern analyses revealed that the microcolonies were not entirely randomly distributed and instead were arranged in a uniform pattern. Detailed time-lapse confocal microscopy at the single-cell level demonstrated that the spatial pattern was the result of an intriguing self-organization: small multicellular clusters moved along the surface to fuse with one another to form microcolonies. This active distribution capability was dependent on environmental factors (carbon source and oxygen) and historical contingency (formation of phenotypic variants). The findings of this study are discussed in the context of species distribution patterns observed in macroecology, and we summarize observations about the processes involved in coadaptation between P. putida and Acinetobacter sp. C6. Our results contribute to an understanding of spatial species distribution patterns as they are observed in nature, as well as the ecology of engineered communities that have the potential for enhanced and sustainable bioprocessing capacity., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Quorum sensing is a process of cell-to-cell communication in which bacteria produce autoinducers as signaling molecules to sense cell density and coordinate gene expression. In the present study, a LuxI-type synthase, AnoI, and a LuxR-type regulator, AnoR, were identified in Acinetobacter nosocomialis, an important nosocomial pathogen, by sequence analysis of the bacterial genome. We found that N-(3-hydroxy-dodecanoyl)-L-homoserine lactone (OH-dDHL) is a quorum-sensing signal in A. nosocomialis. The anoI gene deletion was responsible for the impairment in the production of OH-dDHL. The expression of anoI was almost abolished in the anoR mutant. These results indicate that AnoI is essential for the production of OH-dDHL in A. nosocomialis, and its expression is positively regulated by AnoR. Moreover, the anoR mutant exhibited deficiency in biofilm formation. In particular, motility of the anoR mutant was consistently and significantly abolished compared with that of the wild type. The deficiency in the biofilm formation and motility of the anoR mutant was significantly restored by a functional anoR , indicating that AnoR plays important roles in the biofilm formation and motility. Furthermore, the present study showed that virstatin exerts its effects on the reduction of biofilm formation and motility by inhibiting the expression of anoR . Consequently, the combined results suggest that AnoIR is a quorum-sensing system that plays important roles in the biofilm formation and motility of A. nosocomialis, and virstatin is an inhibitor of the expression of anoR.

We investigated the role of bacterial biofilms in stimulating T helper 17 (Th17) cells in infected organisms. The formation of bacterial biofilms isolated from clinical lavage fluid samples was measured. Th17 cells and interleukin 17 (IL-17) levels in the peripheral blood of healthy individuals, people infected by biofilm bacteria, people infected by non-biofilm bacteria, and in the lavage fluid from people infected by bacteria were determined. Differences in those data were tested using the SPSS 17.0 statistical software. Th17 cells and IL-17 levels in the peripheral blood of biofilm bacteria-infected people, non-biofilm bacteria-infected people, and healthy controls were 0.59 ± 0.18% and 108.8 ± 20.5 pg/mL; 0.58 ± 0.18% and 100.1 ± 20.7 pg/mL; and 0.55 ± 0.17% and 100.0 ± 21.4 pg/mL, respectively; there were no statistically significant differences (P > 0.05). Th17 cells and IL-17 levels in the lavage fluid of biofilm bacteria-infected people and non-biofilm bacteria-infected people were 1.37 ± 0.34% and 157.4 ± 30.8 pg/mL; and 1.11 ± 0.21% and 136.2 ± 24.3 mg/mL, respectively; the differences were statistically significant (P < 0.05). Bacterial biofilms can increase the expression levels of Th17 cells and IL-17 in local infections; this may be the mechanism by which chronic injuries are caused by biofilm infections.

A new strain, named SYF26, was isolated from the Hei He oligotrophic drinking-water reservoir in China. Based on its phenotypic and phylogenetic characteristics, the isolate was identified as a species of genus Acinetobacter. Strain SYF26 was able to grow at low NH₄(+)-N concentrations (5.46 mg l(-1)), and the nitrification rate was 0.064 mg NH₄(+)-N l(-1) h(-1). Low accumulation of nitrate and nitrite was observed throughout the ammonium removal experiment. Strain SYF26 reduced NO₃(-)-N or NO₂(-)-N. Nitrite reductase and periplasmic nitrate reductase were detectable. The putative nitrogen removal process carried out by the strain SYF26 is as follows: NH₄(+)→NH₂OH→NO₂(-)→NO₃(-), then NO₃(-)→NO₂(-)→N₂. Response surface methodology analysis demonstrated that the maximum removal of ammonium occurred under the following conditions: NH₄(+)-N concentration of 22.05 mg l(-1), C/N ratio of 4.31, initial pH of 7.78 and temperature of 29.73 °C, where initial pH and temperature had the largest influence on ammonium removal., (© 2015 The Authors.)

Background: Head louse infestation, which is caused by Pediculus humanus capitis, occurs throughout the world. With the advent of molecular techniques, head lice have been classified into three clades. Recent reports have demonstrated that pathogenic organisms could be found in head lice. Head lice and their pathogenic bacteria in Thailand have never been investigated. In this study, we determined the genetic diversity of head lice collected from various areas of Thailand and demonstrated the presence of Acinetobacter spp. in head lice., Methods: Total DNA was extracted from 275 head louse samples that were collected from several geographic regions of Thailand. PCR was used to amplify the head louse COI gene and for detection of Bartonella spp. and Acinetobacter spp. The amplified PCR amplicons were cloned and sequenced. The DNA sequences were analyzed via the neighbor-joining method using Kimura's 2-parameter model., Results: The phylogenetic tree based on the COI gene revealed that head lice in Thailand are clearly classified into two clades (A and C). Bartonella spp. was not detected in all the samples, whereas Acinetobacter spp. was detected in 10 samples (3.62%), which consisted of A. baumannii (1.45%), A. radioresistens (1.45%), and A. schindleri (0.72%). The relationship of Acinetobacter spp. and the head lice clades showed that Acinetobacter spp. was found in clade A and C., Conclusions: Head lice in Thailand are classified into clade A and B based on the COI gene sequences. Pathogenic Acinetobacter spp. was detected in both clades. The data obtained from the study might assist in the development of effective strategies for head lice control in the future. Detection of pathogenic bacteria in head lice could raise awareness of head lice as a source of nosocomial bacterial infections.

Bacteria frequently exchange metabolites by diffusion through the extracellular environment, yet it remains generally unclear whether bacteria can also use cell-cell connections to directly exchange nutrients. Here we address this question by engineering cross-feeding interactions within and between Acinetobacter baylyi and Escherichia coli, in which two distant bacterial species reciprocally exchange essential amino acids. We establish that in a well-mixed environment E. coli, but likely not A. baylyi, can connect to other bacterial cells via membrane-derived nanotubes and use these to exchange cytoplasmic constituents. Intercellular connections are induced by auxotrophy-causing mutations and cease to establish when amino acids are externally supplied. Electron and fluorescence microscopy reveal a network of nanotubular structures that connects bacterial cells and enables an intercellular transfer of cytoplasmic materials. Together, our results demonstrate that bacteria can use nanotubes to exchange nutrients among connected cells and thus help to distribute metabolic functions within microbial communities.

Strain A2 with algicidal activity against Microcystis aeruginosa was isolated and identified with the genus Acinetobacter on the basis of phenotypic tests and 16S rRNA gene analysis. It was identified with the species Acinetobactor guillouiae by partial rpoB sequence analysis. When 10% (v/v) of the bacterial culture was co-incubated with M. aeruginosa culture, algicidal efficiency reached 91.6% after 7 days. Supernatant of A2 culture showed similar algicidal activity, while the cell pellet had little activity, suggesting that Acinetobacter guillouiae A2 indirectly attacked M. aeruginosa cells by secreting an extracellular algicidal compound, which was characterized as heat-stable. A significant decrease in the microcystin (microcystin-LR) concentration was observed after 10% (v/v) addition of A2 culture. Transcription of three microcystin-related genes (mcyA, mcyD and mcyH) was also found to be inhibited. The algicidal compound 4-hydroxyphenethylamine was obtained by further isolation and purification using various chromatographic techniques. The EC50, 3d and EC50, 7d values of 4-hydroxyphenethylamine against M. aeruginosa were 22.5 and 10.3 mgL(-1), respectively. These results indicate that A. guillouiae strain A2 inhibits growth of M. aeruginosa and degrades microcystin production. The identified compound, 4-hydroxyphenethylamine, has potential for development as a new algicidal formulation or product., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

A taxonomically unique bacterial strain, Acinetobacter sp. A47, has been recovered from several soft tissue samples from a patient undergoing reconstructive surgery owing to a traumatic amputation. The results of 16S rRNA, rpoB, and gyrB gene comparative sequence analyses showed that A47 does not belong to any of the hitherto-known taxa and may represent an as-yet-unknown Acinetobacter species. The recognition of this novel organism contributes to our knowledge of the taxonomic complexity underlying infections caused by Acinetobacter., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Genetic exchange by natural transformation is an important mechanism of horizontal gene transfer in biofilms. Thirty-two biofilm metrics were quantified in a heavily encapsulated Acinetobacter baylyi strain and a miniencapsulated mutant strain, accounting for cellular architecture, extracellular polymeric substances (EPS) architecture, and their combined biofilm architecture. In general, transformation location, abundance, and frequency were more closely correlated to EPS architecture than to cellular or combined architecture. Transformation frequency and transformant location had the greatest correlation with the EPS metric surface area-to-biovolume ratio. Transformation frequency peaked when EPS surface area-to-biovolume ratio was greater than 3 μm(2)/μm(3) and less than 5 μm(2)/μm(3). Transformant location shifted toward the biofilm-bulk fluid interface as the EPS surface area-to-biovolume ratio increased. Transformant biovolume was most closely correlated with EPS biovolume and peaked when transformation occurred in close proximity to the substratum. This study demonstrates that biofilm architecture influences A. baylyi transformation frequency and transformant location and abundance. The major role of EPS may be to facilitate the binding and stabilization of plasmid DNA for cellular uptake., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Acquisition of the extracellular tetracycline (TC) resistance plasmid pAST2 affected host gene expression and phenotype in the oil-degrading soil bacterium, Acinetobacter oleivorans DR1. Whole-transcriptome profiling of DR1 cells harboring pAST2 revealed that all the plasmid genes were highly expressed under TC conditions, and the expression levels of many host chromosomal genes were modulated by the presence of pAST2. The host energy burden imposed by replication of pAST2 led to (i) lowered ATP concentrations, (ii) downregulated expression of many genes involved in cellular growth, and (iii) reduced growth rate. Interestingly, some phenotypes were restored by deleting the plasmid-encoded efflux pump gene tetH, suggesting that the membrane integrity changes resulting from the incorporation of efflux pump proteins also resulted in altered host response under the tested conditions. Alteration of membrane integrity by tetH deletion was shown by measuring permeability of fluorescent probe and membrane hydrophobicity. The presence of the plasmid conferred peroxide and superoxide resistance to cells, but only peroxide resistance was diminished by tetH gene deletion, suggesting that the plasmid-encoded membrane-bound efflux pump protein provided peroxide resistance. The downregulation of fimbriae-related genes presumably led to reduced swimming motility, but this phenotype was recovered by tetH gene deletion. Our data suggest that not only the plasmid replication burden, but also its encoded efflux pump protein altered host chromosomal gene expression and phenotype, which also alters the ecological fitness of the host in the environment.

P. aeruginosa and Acinetobacter spp. are important pathogens associated with late nosocomial pneumonia in hospitalized and institutionalized individuals. The oral cavity may be a major source of these respiratory pathogens, particularly in the presence of poor oral hygiene and periodontal infection. This study investigated the prevalence of P. aeruginosa and Acinetobacter spp. in subgingival biofilm and saliva of subjects with periodontal disease or health. Samples were obtained from 55 periodontally healthy (PH) and 169 chronic periodontitis (CP) patients. DNA was obtained from the samples and detection of P. aeruginosa and Acinetobacter spp. was carried out by multiplex and nested PCR. P. aeruginosa and Acinetobacter spp. were detected in 40% and 45% of all samples, respectively. No significant differences in the distribution of these microorganisms between men and women, subgingival biofilm and saliva samples, patients ≤ 35 and > 35 years of age, and smokers and non-smokers were observed regardless periodontal status (p > 0.05). In contrast, the frequencies of P. aeruginosa and Acinetobacter spp. in saliva and biofilm samples were significantly greater in CP than PH patients (p < 0.01). Smokers presenting P. aeruginosa and high frequencies of supragingival plaque were more likely to present CP than PH. P. aeruginosa and Acinetobacter spp. are frequently detected in the oral microbiota of CP. Poor oral hygiene, smoking and the presence of P. aeruginosa are strongly associated with periodontitis.

Objectives: To determine the in vitro activity of antibiotics, including arbekacin, cefminox, fosfomycin and biapenem which are all still unavailable in India, against Gram-negative clinical isolates., Methods: We prospectively collected and tested all consecutive isolates of Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. from blood, urine and sputum samples between March and November 2012. The minimum inhibition concentration (MIC) of 16 antibiotics was determined by the broth micro-dilution method., Results: Overall 925 isolates were included; 211 E. coli, 207 Klebsiella spp., 153 P. aeruginosa, and 354 Acinetobacter spp. The MIC50 and MIC90 were high for cefminox, biapenem and arbekacin for all pathogens but interpretative criteria were not available. The MIC50 was categorized as susceptible for a couple of antibiotics, including piperacillin/tazobactam, carbapenems and amikacin, for E. coli, Klebsiella spp. and P. aeruginosa. However, for Acinetobacter spp., the MIC50 was categorized as susceptible only for colistin. On the other hand, fosfomycin was the only antibiotic that inhibited 90% of E. coli and Klebsiella spp. isolates, while 90% of P. aeruginosa isolates were inhibited only by colistin. Finally, 90% of Acinetobacter spp. isolates were not inhibited by any antibiotic tested., Conclusion: Fosfomycin and colistin might be promising antibiotics for the treatment of infections due to E. coli or Klebsiella spp. and P. aeruginosa, respectively, in India; however, clinical trials should first corroborate the in vitro findings. The activity of tigecycline should be evaluated, as this is commonly used as last-resort option for the treatment of multidrug-resistant Acinetobacter infections.

Quorum sensing (QS)-dependent biofilm formation and motility were controlled by AqsR in Acinetobacter oleivorans DR1. QS-controlled phenotypes appeared to be inhibited by indole and the aqsR mutant had the same phenotypes. We demonstrated that the turnover rate of AqsR became more rapid without the N-acylhomoserine lactone (AHL) signal, and that indole could increase the expression of many protease and chaperone proteins. The addition of exogenous indole decreased the expression of two AqsR-targeted genes: AOLE&#95;03905 (putative surface adhesion protein) and AOLE&#95;11355 (L-asparaginase). The overexpression of AqsR in Escherichia coli was impossible with the indole treatment. Surprisingly, our [(35)S]methionine pulse-labelling data demonstrated that the stability and folding of AqsR protein decreased in the presence of indole without changing aqsR mRNA expression in E. coli. Interestingly, indole resulted in a loss of TraR-dependent traG expression in an Agrobacterium tumefaciens indicator strain. However, when indole was added after incubation with exogenous AHL, indole could not inhibit the TraR-dependent expression of the traG promoter. This indicated that AHL-bound TraR could be protective against indole, but TraR without AHL could not be active in the presence of indole. Here, we provided evidence for the first time showing that the indole effect on QS-controlled bacterial phenotypes is due to inhibited QS regulator folding and not a reduced QS signal.

In bacterial biofilms, high molecular weight, secreted exopolysaccharides can serve as a scaffold to which additional carbohydrates, proteins, lipids, and nucleic acids adhere, forming the matrix of the developing biofilm. Here we report methods to extract and purify high molecular weight (>15 kDa) exopolysaccharides from biofilms of eight human pathogens, including species of Staphylcococcus, Klebsiella, Acinetobacter, Pseudomonas, and a toxigenic strain of Escherichia coli O157:H7. Glycosyl composition analysis indicated a high total mannose content across all strains with P. aeruginosa and A. baumannii exopolysaccharides comprised of 80-90% mannose, K. pneumoniae and S. epidermidis strains containing 40-50% mannose, and E. coli with ∼10% mannose. Galactose and glucose were also present in all eight strains, usually as the second and third most abundant carbohydrates. N-acetyl-glucosamine and galacturonic acid were found in 6 of 8 strains, while arabinose, fucose, rhamnose, and xylose were found in 5 of 8 strains. For linkage analysis, 33 distinct residue-linkage combinations were detected with the most abundant being mannose-linked moieties, in line with the composition analysis. The exopolysaccharides of two P. aeruginosa strains analyzed were consistent with the Psl carbohydrate, but not Pel or alginate. The S. epidermidis strain had a composition rich in mannose and glucose, which is consistent with the previously described slime associated antigen (SAA) and the extracellular slime substance (ESS), respectively, but no polysaccharide intracellular adhesion (PIA) was detected. The high molecular weight exopolysaccharides from E. coli, K. pneumoniae, and A. baumannii appear to be novel, based on composition and/or ratio analysis of carbohydrates.

Members of the genus Acinetobacter have been the focus recent attention due to both their clinical significance and application to molecular biology. The soil commensal bacterium Acinetobacter baylyi ADP1 has been proposed as a model system for molecular and genetic studies, whereas in a clinical environment, Acinetobacter spp. are of increasing importance due to their propensity to cause serious and intractable systemic infections. Clinically, a major factor in the success of Acinetobacter spp. as opportunistic pathogens can be attributed to their ability to rapidly evolve resistance to common antimicrobial compounds. Whole genome sequencing of clinical and environmental Acinetobacter spp. isolates has revealed the presence of numerous multidrug transporters within the core and accessory genomes, suggesting that efflux is an important host defense response in this genus. In this work, we used the drug-susceptible organism A. baylyi ADP1 as a model for studies into the evolution of efflux mediated resistance in genus Acinetobacter, due to the high level of conservation of efflux determinants across four diverse Acinetobacter strains, including clinical isolates. A single exposure of therapeutic concentrations of chloramphenicol to populations of A. baylyi ADP1 cells produced five individual colonies displaying multidrug resistance. The major facilitator superfamily pump craA was upregulated in one mutant strain, whereas the resistance nodulation division pump adeJ was upregulated in the remaining four. Within the adeJ upregulated population, two different levels of adeJ mRNA transcription were observed, suggesting at least three separate mutations were selected after single-step exposure to chloramphenicol. In the craA upregulated strain, a T to G substitution 12 nt upstream of the craA translation initiation codon was observed. Subsequent mRNA stability analyses using this strain revealed that the half-life of mutant craA mRNA was significantly greater than that of wild-type craA mRNA.

Background: Microbial taxonomy remains a conservative discipline, relying on phenotypic information derived from growth in pure culture and techniques that are time-consuming and difficult to standardize, particularly when compared to the ease of modern high-throughput genome sequencing. Here, drawing on the genus Acinetobacter as a test case, we examine whether bacterial taxonomy could abandon phenotypic approaches and DNA-DNA hybridization and, instead, rely exclusively on analyses of genome sequence data., Results: In pursuit of this goal, we generated a set of thirteen new draft genome sequences, representing ten species, combined them with other publically available genome sequences and analyzed these 38 strains belonging to the genus. We found that analyses based on 16S rRNA gene sequences were not capable of delineating accepted species. However, a core genome phylogenetic tree proved consistent with the currently accepted taxonomy of the genus, while also identifying three misclassifications of strains in collections or databases. Among rapid distance-based methods, we found average-nucleotide identity (ANI) analyses delivered results consistent with traditional and phylogenetic classifications, whereas gene content based approaches appear to be too strongly influenced by the effects of horizontal gene transfer to agree with previously accepted species., Conclusion: We believe a combination of core genome phylogenetic analysis and ANI provides an appropriate method for bacterial species delineation, whereby bacterial species are defined as monophyletic groups of isolates with genomes that exhibit at least 95% pair-wise ANI. The proposed method is backwards compatible; it provides a scalable and uniform approach that works for both culturable and non-culturable species; is faster and cheaper than traditional taxonomic methods; is easily replicable and transferable among research institutions; and lastly, falls in line with Darwin's vision of classification becoming, as far as is possible, genealogical.