BACKGROUND: Acinetobacter nosocomialis (A. nosocomialis) is a glucose non-fermentative, gram-negative bacillus that belongs to the Acinetobacter calcoaceticus-baumannii complex. In recent years, studies have found an increased clinical prevalence of A. nosocomialis. However, given the increasing trend of antibiotic resistance, developing new antibacterial agents is vital. Currently, research regarding bacteriophage therapy against A. nosocomialis is only limited.
METHODS: Two A. nosocomialis bacteriophages, TCUAN1 and TCUAN2, were isolated from sewage. Experiments such as transmission electron microscopy (TEM), host-range analysis, and sequencing were performed to determine their biological and genomic characteristics. TCUAN2 were further subjected to in vivo experiments and their derived-endolysin were cloned and tested against their bacteria host.
RESULTS: Transmission electron microscopy revealed that TCUAN1 and TCUAN2 belong to Myoviridae and Podoviridae, respectively. Both phages show a broad host spectrum and rapid adsorption efficiency. Further biological analysis showed that TCUAN2 possesses a shorter latent period and larger burst size compared to TCUAN1. Because TCUAN2 showed a better antibacterial activity, it was injected into A. nosocomialis-infected mice which resulted in a significant decrease in bacterial load levels in the blood and increased the mice\'s survival. Finally, genomic analysis revealed that the complete nucleotide sequence of TCUAN1 is 49, 691 bps (containing 75 open reading frames) with a G + C content of 39.3%; whereas the complete nucleotide sequence of TCUAN2 is 41, 815 bps (containing 68 open reading frames) with a G + C content of 39.1%. The endolysin gene cloned and purified from TCUAN2 also showed antibacterial activity when used with a chelator EDTA.

BACKGROUND: Acinetobacter calcoaceticus-A. baumannii (ACB) complex pathogens are known for their prevalence in nosocomial infections and extensive antimicrobial resistance (AMR) capabilities. While genomic studies worldwide have elucidated the genetic context of antibiotic resistance in major international clones (ICs) of clinical Acinetobacter spp., not much information is available from Bangladesh. In this study, we analysed the AMR profiles of 63 ACB complex strains collected from Dhaka, Bangladesh. Following this, we generated draft genomes of 15 of these strains to understand the prevalence and genomic environments of AMR, virulence and mobilization associated genes in different Acinetobacter clones.
RESULTS: Around 84% (n = 53) of the strains were extensively drug resistant (XDR) with two showing pan-drug resistance. Draft genomes generated for 15 strains confirmed 14 to be A. baumannii while one was A. nosocomialis. Most A. baumannii genomes fell under three clonal complexes (CCs): the globally dominant CC1 and CC2, and CC10; one strain had a novel sequence type (ST). AMR phenotype-genotype agreement was observed and the genomes contained various beta-lactamase genes including blaOXA-23 (n = 12), blaOXA-66 (n = 6), and blaNDM-1 (n = 3). All genomes displayed roughly similar virulomes, however some virulence genes such as the Acinetobactin bauA and the type IV pilus gene pilA displayed high genetic variability. CC2 strains carried highest levels of plasmidic gene content and possessed conjugative elements carrying AMR genes, virulence factors and insertion sequences.
CONCLUSIONS: This study presents the first comparative genomic analysis of XDR clinical Acinetobacter spp. from Bangladesh. It highlights the prevalence of different classes of beta-lactamases, mobilome-derived heterogeneity in genetic architecture and virulence gene variability in prominent Acinetobacter clonal complexes in the country. The findings of this study would be valuable in understanding the genomic epidemiology of A. baumannii clones and their association with closely related pathogenic species like A. nosocomialis in Bangladesh.

Infections by Acinetobacter species are recognized as a serious global threat due to causing severe disease and their high levels of antibiotic resistance. Acinetobacter baumannii is the most prevalent pathogen in the genus, but infection by Acinetobacter nosocomialis has been reported widely. Diagnosis of patients with A. baumannii infection is often misdiagnosed with other Acinetobacter species, especially A. nosocomialis. This study investigated whether there were significant differences in clinical outcomes between patients infected with A. baumannii versus A. nosocomialis in Northeast Thailand, and to characterize serological responses to infection with these pathogens. The results show that A. baumannii had higher levels of multidrug resistance. Despite this, clinical outcomes for infection with A. baumannii or A. nosocomialis were similar with mortalities of 33% and 36%, respectively. Both pathogens caused community-acquired infections (A. baumannii 35% and A. nosocomialis 29% of cases). Plasma from uninfected healthy controls contained IgG antibody that recognized both organisms, and infected patients did not show a significantly enhanced antibody response from the first week versus 2 weeks later. Finally, the patterns of antigen recognition for plasma IgG were similar for patients infected with A. baumannii or A. nosocomialis infection, and distinct to the pattern for patients infected with non-Acinetobacter. In conclusion, our data revealed that infection with A. nosocomialis was associated with a similarly high level of mortality as infection with A. baumannii, the high rate of community-acquired infection and antibodies in uninfected individuals suggesting that there is significant community exposure to both pathogens. IMPORTANCE Bacterial infections by Acinetobacter species are global threats due to their severity and high levels of antibiotic resistance. A. baumannii is the most common pathogen in the genus; however, infection by A. nosocomialis has also been widely reported but is thought to be less severe. In this study, we have prospectively investigated 48 reported cases of A. baumannii infection in Northeast Thailand, and characterized the serological responses to infection. We found that 14 (29%) of these infections were actually caused by A. nosocomialis. Furthermore, the incidence of antibiotic resistance among A. nosocomialis strains, APACHE II scores, and mortality for patients infected with A. nosocomialis were much higher than published data. Both A. baumannii and A. nosocomialis had unexpectedly mortality rates of over 30%, and both pathogens caused a high rate of community-acquired infections. Importantly, background antibodies in uninfected individuals suggest significant community exposure to both pathogens in the environment.

To analyse the genome sequences of four archival Acinetobacter nosocomialis clinical isolates (designated AC13, AC15, AC21 and AC25) obtained from Terengganu, Malaysia in 2011 to determine their genetic relatedness and basis of antimicrobial resistance.
Antimicrobial susceptibility profiles of the A. nosocomialis isolates were determined by disk diffusion. Genome sequencing was performed using the Illumina NextSeq platform.
The four A. nosocomialis isolates were cefotaxime resistant whereas three isolates (namely, AC13, AC15 and AC25) were tetracycline resistant. The carriage of the blaADC-255-encoded cephalosporinase gene is likely responsible for cefotaxime resistance in all four isolates. Phylogenetic analysis indicated that the three tetracycline-resistant isolates were closely related, with an average nucleotide identity of 99.9%, suggestive of nosocomial spread, whereas AC21 had an average nucleotide identity of 97.9% when compared to these three isolates. The tetracycline-resistant isolates harboured two plasmids: a 13476 bp Rep3-family plasmid of the GR17 group designated pAC13-1, which encodes the tetA(39) tetracycline-resistance gene, and pAC13-2, a 4872 bp cryptic PriCT-1-family plasmid of a new Acinetobacter plasmid group, GR60. The tetA(39) gene was in a 2 001 bp fragment flanked by XerC/XerD recombination sites characteristic of a mobile pdif module. Both plasmids also harboured mobilisation/transfer-related genes.
Genome sequencing of A. nosocomialis isolates led to the discovery of two novel plasmids, one of which encodes the tetA(39) tetracycline-resistant gene in a mobile pdif module. The high degree of genetic relatedness among the three tetracycline-resistant A. nosocomialis isolates is indicative of nosocomial transmission.

Acinetobacter nosocomialis is a prevalent opportunistic pathogen that causes hospital-acquired infections. The increasing threats from A. nosocomialis infections have led to attention from the scientific and medical communities. Metagenomic next-generation sequencing (mNGS) was performed for an exudate specimen collected from an ICU patient with wound infection, followed by sepsis, in Tongji Hospital. Three assembly strategies were employed to recover the genome of A. nosocomialis in the metagenomic sample. Together with publicly available genomes of A. nosocomialis, the features of population genetics and molecular epidemiology were deeply analyzed. A draft genome was reconstructed for the metagenomic strain WHM01, derived from the ST410 A. nosocomialis dominating the microbial community, thereby prompting its highly pathogenic risk, which is associated with infection and persistence. The structure of the bacterial pangenome was characterized, including the 1862 core and 11,815 accessory genes present in the 157 strains. The genetic diversity of the genes coding for the 128 virulence factors assigned to 14 functional categories was uncovered in this nosocomial pathogen, such as the lipooligosaccharide, capsule, type IV pilus, and outer membrane proteins. Our work revealed genomic properties of ST410 A. nosocomialis, which is prevalent in China, and further highlighted that metagenomic surveillance may be a prospective application for evaluating the pathogenic characteristics of the nosocomial opportunistic pathogens.

Carbapenem resistance is increasing among Gram-negative bacteria, including the genus Acinetobacter. This study aimed to characterize, for the first time, the development of carbapenem resistance in clinical isolates of Acinetobacter junii and Acinetobacter nosocomialis conferred by the acquisition of a plasmid-borne blaOXA-24/40 gene and also to characterize the dissemination of this gene between species of Acinetobacter. Carbapenem-resistant A. nosocomialis HUAV-AN66 and A. junii HUAV-AJ77 strains were isolated in the Arnau de Vilanova Hospital (Spain). The genomes were sequenced, and in silico analysis were performed to characterize the genetic environment and the OXA-24/40 transmission mechanism. Antibiotic MICs were determined, and horizontal transfer assays were conducted to evaluate interspecies transmission of OXA-24/40. Carbapenems MICs obtained were ≥64 mg/L for HUAV-AN66 and HUAV-AJ77. Genome analysis revealed the presence in both strains of a new plasmid, designated pHUAV/OXA-24/40, harboring the carbapenem-resistance gene blaOXA-24/40 and flanked by sequences XerC/XerD. pHUAV/OXA-24/40 was successfully transferred from A. nosocomialis and A. junii to a carbapenem-susceptible A. baumannii strain, thus conferring carbapenem resistance. A second plasmid (pHUAV/AMG-R) was identified in both clinical isolates for the successful horizontal transfer of pHUAV/OXA-24/40. blaOXA-24/40-carrying plasmids of the GR12 group and showing high identity with pHUAV/OXA-24/40 were identified in at least 8 Acinetobacter species. In conclusion the carbapenemase OXA-24/40 is described for the first time in A. nosocomialis and A. junii. In both isolates the blaOXA-24/40 gene was located in the GR12 pHUAV/OXA-24/40 plasmid. GR12 plasmids are implicated in the dissemination and spread of carbapenem resistance among Acinetobacter species. IMPORTANCE Acinetobacter baumannii is one of the most relevant pathogens in terms of antibiotic resistance. The main resistance mechanisms are the carbapenem-hydrolyzing class D β-lactamases (CHDLs), especially OXA-23 and OXA-24/40. In addition to A. baumannii, there are other species within the genus Acinetobacter, which in general exhibit much lower resistance rates. In this work we characterize for the first time two clinical isolates of Acinetobacter nosocomialis and Acinetobacter junii, isolated in the same hospital, carrying the carbapenemase OXA-24/40 and displaying high resistance rates to carbapenems. By means of bioinformatics analysis we have also been able to characterize the mechanism by which this carbapenemase is horizontally transferred interspecies of Acinetobacter spp. The dissemination of carbapenemase OXA-24/40 between non-baumannii Acinetobacter species is concerning since it prevents the use of most β-lactam antibiotics in the fight against these resistant isolates.

Infections caused by extensively drug-resistant (XDR) Acinetobacter nosocomialis have become a challenging problem. The frequent use of colistin as the last resort drug for XDR bacteria has led to the emergence of colistin-resistant A. nosocomialis (ColRAN) in hospitals. The mechanism of colistin resistance in A. nosocomialis remains unclear. This study aimed to investigate the mechanisms underlying colistin resistance in clinical ColRAN isolates. We collected 36 A. nosocomialis isolates from clinical blood cultures, including 24 ColRAN and 12 colistin-susceptible A. nosocomialis (ColSAN). The 24 ColRAN isolates clustered with ST1272 (13), ST433 (eight), ST1275 (two), and ST410 (one) by multilocus sequence typing. There was a positive relationship between pmrCAB operon expression and colistin resistance. Further analysis showed that colistin resistance was related to an amino acid substitution, Ser253Leu in PmrB. By introducing a series of recombinant PmrB constructs into a PmrB knockout strain and protein structural model analyses, we demonstrated that the association between Ser253Leu and Leu244 in PmrB was coupled with colistin resistance in ColRAN. To the best of our knowledge, this is the first study demonstrating that the key amino acid Ser253Leu in PmrB is associated with overexpression of the pmrCAB operon and hence colistin resistance. This study provides insight into the mechanism of colistin resistance in A. nosocomialis.

We identified nine Verona integron-encoded metallo-β-lactamase-2 (VIM-2)-producing Acinetobacter nosocomialis (n = 8) and Acinetobacter seifertii (n = 1) isolates from South Korea and performed whole-plasmid sequencing for two A. nosocomialis isolates and one A. seifertii isolate. Genotyping, antibiotic resistance profiles, and whole plasmid sequences indicated clonal dissemination of the eight VIM-2-producing A. nosocomialis isolates. The plasmid-bearing blaVIM-2 in the A. seifertii isolate differed from those in the A. nosocomialis isolates.

Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes blaNDM-1 and blaOXA-58 in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The blaNDM-1 gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas blaOXA-58 was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.

The present study was aimed to investigate the antibiofilm activity of 5-hydroxymethylfurfural against Acinetobacter baumanni and Vellar estuary isolates v3 (Acinetobacter nosocomialis). The biofilm inhibitory concentration (BIC) of 5HMF against A. baumannii and v3 (A. nosocomialis) was found to be 100 µg/ml) exhibited non-bactericidal concentration-dependent antibiofilm activities against Acinetobacter species. The present study found that 5HMF treatment is very effective in the initial stage of A. baumannii biofilms and it significantly disrupted the mature biofilms. Moreover, 5HMF treatment inhibited the extracellular polymeric substances (EPS), including polysaccharides and proteins production. Results from gene expression and in vitro assays further demonstrated the 5HMF treatment downregulated the expression of bfmR, bap, csuA/B, ompA and katE virulence genes, which consistently affects biofilm formation and its mediated virulence property. The present study suggests that 5HMF unveil its antibiofilm activity by interfering initial biofilm formation and suppressing the virulence regulator genes in A. baumannii. Further studies are required to explore the 5HMF mode of action responsible for the antibiofilm activity.