A rapid bacterial pathogen and antimicrobial resistance diagnosis workflow using Oxford nanopore adaptive sequencing method.

Metagenomic sequencing analysis (mNGS) has been implemented as an alternative approach for pathogen diagnosis in recent years, which is independent of cultivation and is able to identify all potential antibiotic resistance genes (ARGs). However, current mNGS methods have to deal with low amounts of prokaryotic deoxyribonucleic acid (DNA) and high amounts of host DNA in clinical samples, which significantly decrease the overall microbial detection resolution. The recently released nanopore adaptive sampling (NAS) technology facilitates immediate mapping of individual nucleotides to a given reference as each molecule is sequenced. User-defined thresholds allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. We developed a metagenomics workflow for ultra-sensitive diagnosis of bacterial pathogens and ARGs from clinical samples, which is based on the efficient selective 'human host depletion' NAS sequencing, real-time species identification and species-specific resistance gene prediction. Our method increased the microbial sequence yield at least 8-fold in all 21 sequenced clinical Bronchoalveolar Lavage Fluid (BALF) samples (4.5 h from sample to result) and accurately detected the ARGs at species level. The species-level positive percent agreement between metagenomic sequencing and laboratory culturing was 100% (16/16) and negative percent agreement was 100% (5/5) in our approach. Further work is required for a more robust validation of our approach with large sample size to allow its application to other infection types.

Biofilm formation in erythromycin-resistant Staphylococcus aureus and the relationship with antimicrobial susceptibility and molecular characteristics.

PURPOSE: In this study, we aimed to investigate biofilm formation characteristics in clinical Staphylococcus aureus (S. aureus) isolates with erythromycin (ERY) resistance from China and further analyze their correlations with antimicrobial susceptibility and molecular characteristics. METHODOLOGY: A total of 276 clinical isolates of ERY-resistant S. aureus, including 142 methicillin-resistant S. aureus (MRSA) strains and 134 methicillin-susceptible S. aureus (MSSA) strains, were retrospectively collected in China. Biofilms were determined by crystal violet staining and ERY resistance genes (ermA, ermB and ermC) were detected by polymerase chain reaction. Inducible clindamycin resistance was examined by D test and multilocus sequence typing, and clonal complexes (CCs) based on housekeeping genes were further determined. RESULTS: The frequency of biofilm formation among ERY-resistant S. aureus was 40.9% (113/276) in total and no significant difference was found for the frequency of biofilm formation between ERY-resistant MRSA and ERY-resistant MSSA (44.4% vs 37.3%, P > 0.05). In ERY-resistant MRSA isolates, the frequency of biofilm formation in ermA-positive, gentamicin-resistant and ciprofloxacin-resistant isolates was higher than that in ermA-negative, gentamicin-sensitive and ciprofloxacin-sensitive isolates, respectively (63.9% vs 23.6%, P < 0.01; 60.3% vs 27.5%, P < 0.01; 65.2% vs 26.3%, P < 0.01). In addition, tetracycline resistance facilitated biofilm formation in both ERY-resistant MRSA and MSSA and the frequency of biofilm formation in CC239- or CC7S. aureus isolates with ERY resistance was significantly higher compared with that in CC59S. aureus (both P < 0.01). CONCLUSION: The ermA gene, and gentamicin, ciprofloxacin and tetracycline resistance facilitate biofilm formation in ERY-resistant MRSA isolates and, moreover, ERY-resistant S. aureus isolates with positive biofilm formation exhibited clonality clustering regarding CC239 and CC7.

In vitro-induced erythromycin resistance facilitates cross-resistance to the novel fluoroketolide, solithromycin, in Staphylococcus aureus.

The aim of this study was to determine whether in vitro induced erythromycin resistance facilitates the cross-resistance to the novel fluoroketolide, solithromycin, in Staphylococcus aureus. Four strains of methicillin-susceptible S. aureus strains S2, S3, S5 and S7 were successfully induced to establish erythromycin-resistant strains by continuous in vitro culture with erythromycin. Mutations at drug binding sites were shown to increase the minimal inhibitory concentrations for ketolides, including telithromycin and the novel compound solithromycin, but did not increase for lincosamides, chloramphenicols or oxazolidinones. In S2-, S5- and S7-derived strains, L22 protein mutations occurred first, resulting in a low level of cross-resistance to ketolides (≤4 µg/mL). The L4 protein mutations were dependent on the L22 protein, resulting in high-level cross-resistance to ketolides (≥8 µg/mL). In S3-derived strains, high levels of cross-resistance occurred concurrently in the 23S rRNA domains II/V and the L22 protein. Hence, long-term exposure of erythromycin results in resistance to ketolides in S. aureus through drug binding site mutations. These results demonstrate that since erythromycin has been used clinically for a long time, it is necessary to carefully evaluate the rewards and risks when prescribing solithromycin for the treatment of infectious diseases.

Effect of tedizolid on clinical Enterococcus isolates: in vitro activity, distribution of virulence factor, resistance genes and multilocus sequence typing.

Enterococcal infections have become one of the most challenging nosocomial problems. Tedizolid, the second oxazolidinone, is 4-fold to 8-fold more potent in vivo and in vitro than linezolid against enterococci. However, the characteristics of tedizolid related to enterococci isolates in China remain elusive. The aim of this study was to evaluate in vitro activity of tedizolid against enterococcal isolates from patients with infections at a teaching hospital in China and to investigate the correlations between in vitro tedizolid activity against enterococci and the distribution of multilocus sequence types (MLST), resistance genes and virulence factors. A total of 289 non-duplicate Enterococcus faecalis strains and 68 E. faecium strains were isolated. Tedizolid inhibited 95.24% of all enterococcal isolates with an MIC ≤ 0.5µg/ml. Seventeen E. faecalis strains had an MIC > 0.5 µg/ml, and all E. faecium were inhibited at MIC ≤ 0.5 µg/ml. The proportion of tedizolid non-susceptible E. faecalis strains with optrA genes was higher than that among tedizolid-susceptible strains. Tedizolid exhibited good in vitro activity against all E. faecium strains, including multidrug-resistant E. faecium carrying tet(M), tet(L), tet(U),erm(A), erm(B) and erm(C) genes. In summary, tedizolid has an advantage (higher sensitivity rate) compared to linezolid among enterococci, except for isolates expressing the plasmid-encoded optrA gene.

Rhabdomyolysis, lactic acidosis, and multiple organ failure during telbivudine treatment for hepatitis B: a case report and review of the literature.

BACKGROUND: Telbivudine can cause severe side effects, including myositis, neuritis, rhabdomyolysis, and lactic acidosis. However, reported cases of telbivudine leading to multiple organ failure are rare. Here, we report a case of telbivudine-induced severe polymyositis, lactic acidosis, and multiple organ failure. CASE PRESENTATION: A 30-year-old Chinese man with hepatitis B virus infection received antiviral treatment with 600 mg of telbivudine daily for more than 11 months. He developed progressive weakness and myalgia, and subsequently experienced palpitations, chest tightness, lethargy, hypotension, and hypoxemia. Blood tests showed markedly elevated levels of alanine aminotransferase (955 U/L), aspartate aminotransferase (1375 U/L), blood urea nitrogen (14.9 mmol/L), creatine kinase (peak at 8050 U/L), and blood lactate (>20.0 mmol/L). His symptoms improved after continuous renal replacement therapy and short-term methylprednisolone treatment. Hyperbaric oxygen therapy, physical therapy, and rehabilitation for more than 2 months led to recovery of muscle strength to the normal range. CONCLUSIONS: We conclude that continuous renal replacement and steroid therapies play key roles in stabilizing telbivudine-induced severe rhabdomyolysis, lactic acidosis, and multiple organ failure. Hyperbaric oxygen, physical therapy, and rehabilitation may aid in functional recovery after the acute phase of lactic acidosis and organ failure.