Genome characterization of the novel lytic phage vB_AbaAut_ChT04 and the antimicrobial activity of its lysin peptide against Acinetobacter baumannii isolates from different time periods.

Acinetobacter baumannii is an important opportunistic pathogen, usually associated with immunocompromised individuals with a prolonged period of stay in a hospital. Currently, the incidence of multi-drug resistant A. baumannii (MDR-AB) and extensively drug-resistant A. baumannii (XDR-AB) is increasing rapidly in Thailand, mirroring the trend worldwide. Novel therapeutic approaches for the treatment of A. baumannii infection using bacteriophages are being evaluated, and the use of phage-derived peptides is being tested as alternative approach to fighting infection. In this study, we isolated and determined the biological features of a lytic A. baumannii phage called vB_AbaAut_ChT04 (vChT04). The vChT04 phage was classified as a member of the family Autographiviridae of the class Caudoviricetes. It showed a short latent period (10 min) and a large burst size (280 PFU cell-1), and it was able to infect 52 out of 150 clinical MDR-AB strains tested (34.67%). Most of the phage-sensitive strains were A. baumannii strains that had been isolated during the same year that the phage was isolated. The phage showed activity across a broad pH (pH 5.0-8.0) and temperature (4-37°C) range. Whole-genome analysis revealed that the vChT04 genome comprises 41,158 bp with a 39.3% GC content and contains 48 open reading frames (ORFs), 28 of which were assigned putative functions based on homology to previously identified phage genes. Comparative genomic analysis demonstrated that vChT04 had the highest similarity to phage vB_AbaP_WU2001, which was isolated in the southern part of Thailand. An endolysin gene found in the vChT04 genome was used to synthesize an antimicrobial peptide (designated as PLysChT04) and its antimicrobial activity was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The MIC and MBC values of peptide PLysChT04 against MDR-AB and XDR-AB were 312.5-625 µg/mL, and it was able to inhibit both phage-susceptible and phage-resistant isolates collected over different time periods. PLysChT04 showed good efficacy in killing drug-resistant A. baumannii and other bacterial strains, and it is a promising candidate for development as an alternative therapeutic agent targeting A. baumannii infections.

Genomic relatedness and dissemination of bla NDM-5 among Acinetobacter baumannii isolated from hospital environments and clinical specimens in Thailand.

Background: Acinetobacter baumannii (A. baumannii) is an important cause of nosocomial infection, especially in intensive care units (ICUs). It has the propensity to tolerate various environments and multiple classes of antibiotics. Our study aimed to characterize the comparative genomes of A. baumannii from hospital environments and clinical isolates. Methods: Clinical and environmental A. baumannii isolates were collected from a university hospital. Antibiotic susceptibility testing was performed, antibiotic resistance genes (ARGs) were characterized, and repetitive element palindromic-PCR (rep-PCR) typing was performed. Eight representative A. baumannii isolated from environmental and clinical samples from the same wards were selected for whole-genome sequencing (WGS) using the Illumina platform. Results: A total of 106 A. baumannii isolates were obtained from 312 hospital environmental samples. A high percentage of samples with A. baumannii colonization were detected from AMBU bags (77.9%), followed by bedrails (66.7%) and suction tubes (66.7%). We found that 93.4% of the environmental isolates were multidrug-resistant A. baumannii (MDRAB), and 44.7% were extremely drug-resistant A. baumannii (XDRAB). bla OXA-23 bla NDM, and bla OXA-58 were present in 80.2%, 78.3%, and 0.9% of all isolates, respectively. Sixty-one A. baumannii isolates were collected from patient specimens in the same ward. Among all A. baumannii clinical isolates, MDRAB and XDRAB accounted for 82% and 55.7%, respectively. The most dominant ARGs identified was bla OXA-23 (80.3%), followed by bla NDM (55.7%). The genetic diversity of all isolates using rep-PCR could be divided into 33 genotypes. The genome size of eight A. baumannii ranged from 3.78-4.01 Mb. We found six of eight strains to be bla NDM-5-harboring A. baumannii. Mobile genetic elements (MGEs), such as integron1 (intl1), located upstream of bla NDM-5 were observed. The phylogenomic relationship of the core and pan genomes as well as the single nucleotide polymorphism (SNP) count matrix revealed the genetic similarity of A. baumannii environmental and clinical strains obtained from the same ward. Conclusion: This study confirmed that A. baumannii colonized in hospital environments were the main reservoir of nosocomial infection and provides critical information to guide the control of A. baumannii infection.

Antibacterial activity of vB_AbaM_PhT2 phage hydrophobic amino acid fusion endolysin, combined with colistin against Acinetobacter baumannii.

Phage lytic enzymes are promising antimicrobial agents. In this study, an endolysin derived from vB_AbaM_PhT2 (vPhT2), was identified. This endolysin represented the conserved lysozyme domain. Recombinant endolysin (lysAB- vT2) and hydrophobic fusion endolysin (lysAB-vT2-fusion) were expressed and purified. Both endolysins showed lytic activity against bacterial crude cell wall of Gram-negative bacteria. The MIC of lysAB-vT2-fusion was 2 mg/ml corresponding to 100 µM, while the MIC of lysAB-vT2 was more than 10 mg/ml (400 µM). Combination of lysAB-vT2-fusion with colistin, polymyxin B or copper was synergistic against A. baumannii (FICI value as 0.25). Antibacterial activity of lysAB-vT2-fusion plus colistin at the fractional inhibitory concentrations (FICs) revealed that it can inhibit Escherichia coli, Klebsiella pneumoniae and various strains of extremely drug-resistant A. baumannii (XDRAB) and phage resistant A. baumannii. The lysAB- vT2-fusion still retained its antibacterial activity after incubating the enzyme at 4, 20, 40 and 60 °C for 30 min. The lysAB-vT2-fusion could inhibit the mature biofilm, and incubation of lysAB-vT2-fusion with T24 human cells infected with A. baumannii led to a partial reduction of LDH release from T24 cells. In summary, our study highlights the antimicrobial ability of engineered lysAB-vT2-fusion endolysin, which can be applied for the control of A. baumannii infection.

Insights into mobile genetic elements and the role of conjugative plasmid in transferring aminoglycoside resistance in extensively drug-resistant Acinetobacter baumannii AB329.

Acinetobacter baumannii is a major cause of nosocomial infection, and the incidence of extensively drug-resistant A. baumannii (XDRAB) infections has dramatically increased worldwide. In this study, we aimed to explore the complete genome sequence of XDRAB 329, ST1166/98 (Oxford/Pasteur), which is an outbreak clone from a hospital in Thailand. Whole-genome sequencing (WGS) was performed using short-read Illumina and long-read PacBio sequencing, and a conjugation assay of its plasmid was performed. The complete genome sequence of A. baumannii AB329 revealed a circular chromosome 3,948,038 bp in length with 39% GC content. Antibiotic resistance genes (ARGs), including beta-lactam resistance (bla OXA-51, bla ADC-25, bla OXA-23, bla TEM-1D), aminoglycoside resistance (aph(3')-Ia, aph(3″)-Ib, aph(6)-Id, armA), tetracycline resistance (tet(B), tet (R)), macrolide resistance (mph(E), msr(E)), and efflux pumps, were found. Mobile genetic elements (MGEs) analysis of A. baumannii AB329 revealed two plasmids (pAB329a and pAB329b), three prophages, 19 genomic islands (GIs), and 33 insertion sequences (ISs). pAB329a is a small circular plasmid of 8,731 bp, and pAB329b is a megaplasmid of 82,120 bp. aph(3')-VIa was detected in pAB329b, and a major facilitator superfamily (MFS) transporter was detected in the prophage. Acinetobacter baumannii resistance island 4 (AbaR4) harboring tetracycline and aminoglycoside resistance was detected in the genome of A. baumannii AB329. pAB329b, which belongs to Rep-type GR6 (plasmid lineage LN_1), is a conjugative plasmid with the ability to transfer an aminoglycoside resistance gene to sodium azide-resistant A. baumannii. This study provides insights into the features of the MGEs of XDRAB, which are the main reservoir and source of dissemination of ARGs.

Genomic analysis uncovers laccase-coding genes and biosynthetic gene clusters encoding antimicrobial compounds in laccase-producing Acinetobacter baumannii.

Laccases are multicopper oxidase family enzymes that can oxidize various substrates. In this study, we isolated laccase-producing Acinetobacter spp. from the environment, and one isolate of laccase-producing Acinetobacter baumannii, designated NI-65, was identified. The NI-65 strain exhibited constitutive production of extracellular laccase in a crude extract using 2,6-dimethoxyphenol as a substrate when supplemented with 2 mM CuSO4. Whole-genome sequencing of the NI-65 strain revealed a genome size of 3.6 Mb with 3,471 protein-coding sequences. The phylogenetic analysis showed high similarity to the genome of A. baumannii NCIMB8209. Three laccase proteins, PcoA and CopA, that belong to bacterial CopA superfamilies, and LAC-AB, that belongs to the I-bacterial bilirubin oxidase superfamily, were identified. These proteins were encoded by three laccase-coding genes (pcoA, copA, and lac-AB). The lac-AB gene showed a sequence similar to that of polyphenol oxidase (PPO). Gene clusters encoding the catabolized compounds involved in the utilization of plant substances and secondary metabolite biosynthesis gene clusters encoding antimicrobial compounds were identified. This is the first report of whole-genome sequencing of laccase-producing A. baumannii, and the data from this study help to elucidate the genome of A. baumannii to facilitate its application in synthetic biology for enzyme production.