In order to understand the role of biofilm in the emergence of antibiotic resistance, a total of 104 clinical Acinetobacter baumannii strains were investigated for their biofilm-forming capacities and genes associated with biofilm formation. Selected biofilm-formers were tested for antibiotic susceptibilities when grown in biofilm phase. Reversibility of antibiotic susceptibility in planktonic cells regrown from biofilm were investigated. We found 59.6% of the strains were biofilm-formers, among which, 66.1% were non-multidrug resistant (MDR) strains. Presence of virulence genes bap, csuE, and abaI was significantly associated with biofilm-forming capacities. When strains were grown in biofilm state, the minimum biofilm eradication concentrations were 44, 407, and 364 times higher than the minimum bactericidal concentrations (MBC) for colistin, ciprofloxacin, and imipenem, respectively. Persisters were detected after treating the biofilm at 32–256 times the MBC of planktonic cells. Reversibility test for antibiotic susceptibility showed that biofilm formation induced reversible antibiotic tolerance in the non-MDR strains but a higher level of irreversible resistance in the extensively drug-resistant (XDR) strain. In summary, we showed that the non-MDR strains were strong biofilm-formers. Presence of persisters in biofilm contributed to the reduced antibiotic susceptibilities. Biofilm-grown Acinetobacter baumannii has induced antibiotic tolerance in non-MDR strains and increased resistance levels in XDR strains. To address the regulatory mechanisms of biofilm-specific resistance, thorough investigations at genome and transcription levels are warranted.

Antibiotics at suboptimal doses promote biofilm formation and the development of antibiotic resistance. The underlying molecular mechanisms, however, were not investigated. Here, we report the effects of sub-minimum inhibitory concentrations (sub-MICs) of imipenem and colistin on genes associated with biofilm formation and biofilm-specific antibiotic resistance in a multidrug-tolerant clinical strain of Acinetobacter baumannii Sequence Type (ST) 1894. Comparative transcriptome analysis was performed in untreated biofilm and biofilm treated with sub-MIC doses of imipenem and colistin. RNA sequencing data showed that 78 and 285 genes were differentially expressed in imipenem and colistin-treated biofilm cells, respectively. Among the differentially expressed genes (DEGs), 48 and 197 genes were upregulated exclusively in imipenem and colistin-treated biofilm cells, respectively. The upregulated genes included those encoding matrix synthesis (pgaB), multidrug efflux pump (novel00738), fimbrial proteins, and homoserine lactone synthase (AbaI). Upregulation of biofilm-associated genes might enhance biofilm formation when treated with sub-MICs of antibiotics. The downregulated genes include those encoding DNA gyrase (novel00171), 30S ribosomal protein S20 (novel00584), and ribosome releasing factor (RRF) were downregulated when the biofilm cells were treated with imipenem and colistin. Downregulation of these genes affects protein synthesis, which in turn slows down cell metabolism and makes biofilm cells more tolerant to antibiotics. In this investigation, we also found that 5 of 138 small RNAs (sRNAs) were differentially expressed in biofilm regardless of antibiotic treatment or not. Of these, sRNA00203 showed the highest expression levels in biofilm. sRNAs regulate gene expression and are associated with biofilm formation, which may in turn affect the expression of biofilm-specific antibiotic resistance. In summary, when biofilm cells were exposed to sub-MIC doses of colistin and imipenem, coordinated gene responses result in increased biofilm production, multidrug efflux pump expression, and the slowdown of metabolism, which leads to drug tolerance in biofilm. Targeting antibiotic-induced or repressed biofilm-specific genes represents a new strategy for the development of innovative and effective treatments for biofilm-associated infections caused by A. baumannii.

Escherichia coli sequence type 405 is an emerging antibiotic-resistant clonal group associated with the global dissemination of extended-spectrum β-lactamase-producing E. coli. In this study, we report the genome assembly and characterization of a uropathogenic E. coli ST405 strain, SZESBLEC201, based on long and short reads obtained from the Nanopore and Illumina sequencing platforms, respectively. Whole-genome sequencing revealed that SZESBLEC201 harbors a 5,020,403 bp chromosome and three plasmids, namely, pSZESBLEC201-1, pSZESBLEC201-2, and pSZESBLEC201-3. pSZESBLEC201-1 (111,621 bp) belongs to the IncFIA-FIB type and harbors bla_CTX-M-15. However, this plasmid does not harbor conjugative transfer-associated genes, rendering pSZESBLEC201-1 unable to be conjugatively transferred. pSZESBLEC201-2 (95,138 bp) is a phage-like plasmid that shows a strong genome synteny with Escherichia phage P1 but with the absence of mobile genetic elements and some regulatory genes. pSZESBLEC201-3 (92,865 bp) belongs to the IncI1 type and carries bla_CTX-M-24. In contrast to pSZESBLEC201-1, pSZESBLEC201-3 retains its full active conjugation machinery and can be transferred via conjugation. The genetic features of the genome show that the SZESBLEC201 has a unique virulence pattern compared with genetically similar strains found in the same country (China). The plasmid backbones exhibit a high degree of similarity to those of geographically distant isolates, highlighting the global spread of bla_CTX-M genes and the genome plasticity of this clonal group. The coexistence of two bla_CTX-M variants in the same strain increases the risk of the emergence of new bla_CTX-M variants. Further studies on phage-like plasmids are necessary to provide insights into their biological activities and clinical significance.

Small RNAs (sRNAs) are post-transcriptional regulators of many biological processes in bacteria, including biofilm formation and antibiotic resistance. The mechanisms by which sRNA regulates the biofilm-specific antibiotic resistance in Acinetobacter baumannii have not been reported to date. This study aimed to investigate the influence of sRNA00203 (53 nucleotides) on biofilm formation, antibiotic susceptibility, and expression of genes associated with biofilm formation and antibiotic resistance. The results showed that deletion of the sRNA00203-encoding gene decreased the biomass of biofilm by 85%. Deletion of the sRNA00203-encoding gene also reduced the minimum biofilm inhibitory concentrations for imipenem and ciprofloxacin 1024- and 128-fold, respectively. Knocking out of sRNA00203 significantly downregulated genes involved in biofilm matrix synthesis (pgaB), efflux pump production (novel00738), lipopolysaccharide biosynthesis (novel00626), preprotein translocase subunit (secA) and the CRP transcriptional regulator. Overall, the suppression of sRNA00203 in an A. baumannii ST1894 strain impaired biofilm formation and sensitized the biofilm cells to imipenem and ciprofloxacin. As sRNA00203 was found to be conserved in A. baumannii, a therapeutic strategy targeting sRNA00203 may be a potential solution for the treatment of biofilm-associated infections caused by A. baumannii. To the best of the authors' knowledge, this is the first study to show the impact of sRNA00203 on biofilm formation and biofilm-specific antibiotic resistance in A. baumannii.

The ongoing emergence and spread of drug-resistant pathogens necessitate urgent solutions. Natural products from bacterial sources are recognized as a promising source of antibiotics. This study aimed to isolate and characterize soil microorganisms from extremely hot environments and to screen their secondary metabolites for antibacterial activity. Bacterial isolates were identified using standard culture techniques. Primary and secondary screenings for antimicrobial activity were conducted using the Modified Kirby-Bauer antibiotic susceptibility test against five bacterial species. Based on the efficacy of antimicrobial activity against these target pathogens, the isolate Pseudomonas sp. strain ASTU00105 was selected for further characterization through whole genomic sequencing. Secondary metabolites were analyzed using GC–MS, and antioxidant activities were also evaluated. A total of 76 isolates were identified, and their secondary metabolites were tested against Escherichia coli, Salmonella typhi, Acinetobacter baumannii, Staphylococcus aureus, Streptococcus pyogenes, and Candida albicans. Seventeen isolates (22.37%) exhibited antimicrobial activity. Isolate ASTU00105 exhibited the highest activity against all the test organisms and was selected for further analysis. Whole-genome sequencing using the Nanopore MinION sequencer revealed that strain ASTU00105 belonged to the genus Pseudomonas with the highest similarity (95.97%) to Pseudomonas stutzeri, and designated as Pseudomonas sp. strain ASTU00105. Upon Average Nucleotide Identity (ANI) analysis, the strain exhibited 87.81% sequence similarity with genes of the closest type strain, suggesting its novelty and distinctiveness within the Pseudomonas genus. The genomic analysis of the isolated strain revealed 6 biosynthetic gene cluster (BGC) genes dispersed throughout the entire genome, which are implicated in the synthesis of antimicrobial secondary metabolites. The major chemical compounds detected in the EtAc extracts as detected by gas chromatography-mass spectrometry (GC–MS) were phenol, 2,5-bis (1,1-dimethylethyl) (36.6%), followed by 1,2-Benzenedicarboxylic acid, diethyl ester (12.22%), Eicosane (9.71%), Dibutyl phthalate (3.93%), and 1-Dodecanol (2.34%). Pseudomonas sp. strain ASTU00105 exhibited the greatest potential for producing secondary metabolites with significant antimicrobial activity.

The study was carried out with the aim to isolate Staphylococcus aureus from camel and goat raw milk and determine their antimicrobial susceptibility pattern. 204 raw milk samples were collected from randomly selected lactating camels (n=62) and lactating goats (n=142) in live-stock producing pastoralists’ areas of Somali region for isolation and identification of S. aureus. Antibiotic susceptibility tests were performed on all S. aureus isolates to 16 antibiotics by Kirby-Bauer disk diffusion method. Twenty three (11.27% of the total) S. aureus strains were isolated. Four (6.45%) strains were isolated from camel raw milk samples and 19 (13.34%) from goat raw milk samples. S. aureus isolates showed resistance to Nalidixic acid, Polymixin B, and Penicillin G. S. aureus isolates were sensitive to vancomycin, ciprofloxacin, cefoxitin, cephalotin, gentamycin, Doxycycline, kanamycin, trimethoprim-sulfamethoxazole, chloramphenicol, norfloxacin, and erythromycin. Multi drug resistance was detected in 69.2% of the isolates. The present study has demonstrated the existence of alarmingly high level of multiple antimicrobial resistances of S. aureus among camel and goat milks. Key words: Staphylococcus aureus, camel and goat milk, antimicrobial susceptibility test, Ethiopia.