Generation and characterisation of P. falciparum parasites with a G358S mutation in the PfATP4 Na+ pump and clinically relevant levels of resistance to some PfATP4 inhibitors

Small-molecule inhibitors of PfATP4, a Plasmodium falciparum protein that is believed to pump Na+ out of the parasite while importing H+, are on track to become much-needed new antimalarial drugs. The spiroindolone cipargamin is poised to become the first PfATP4 inhibitor to reach the field, having performed strongly in Phase 1 and 2 clinical trials. Previous attempts to generate cipargamin-resistant parasites in the laboratory have yielded parasites with reduced susceptibility to the drug; however, the highest 50% inhibitory concentration reported to date is 24 nM. Here, we show that P. falciparum parasites can acquire a clinically-significant level of resistance to cipargamin that enables them to withstand micromolar concentrations of the drug. Independent experiments to generate high-level cipargamin resistance using different protocols and strains led to the same change each time - a G358S mutation in PfATP4. Parasites with this mutation showed high-level resistance not only to cipargamin, but also to the dihydroisoquinolone (+)-SJ733. However, for certain other (less clinically advanced) PfATP4-associated compounds the G358S mutation in PfATP4 conferred only moderate resistance or no resistance. The G358S mutation in PfATP4 did not affect parasite susceptibility to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in the Toxoplasma gondii ATP4 homologue (G419S), decreased the sensitivity of the Na+-ATPase activity of ATP4 to inhibition by cipargamin and (+)-SJ733, and decreased the sensitivity of parasites expressing these ATP4 mutations to disruption of parasite Na+ regulation by cipargamin- and (+)-SJ733. The G358S mutation in PfATP4 reduced the affinity of the protein for Na+ and was associated with an increase in the parasite's resting cytosolic Na+ concentration; however, no significant defect in parasite growth rate was observed. Our findings suggest that codon 358 in pfatp4 should be monitored closely in the field as a molecular marker for cipargamin resistance, and that PfATP4 inhibitors in clinical development should be tested for their activity against PfATP4G358S parasites.

Mupirocin Resistance of Staphylococcus aureus in Clinical Isolates of National Hospital and in the Nasal Carriage of Healthy Undergraduates in Colombo, Sri Lanka

Introduction and Objectives : Mupirocin resistance in Staphylococcus aureus is increasingly reported in many parts of the world. This study was conducted with the objective of describing high-level and low-level mupirocin resistance of S. aureus in clinical isolates and nasal carriage. Materials and Methods : A descriptive study was conducted including 45 nasal isolates of S. aureus collected from healthy university students in Colombo and 249 clinical isolates of S. aureus from the patient specimens in National Hospital of Sri Lanka. All of the confirmed S. aureus strains were tested for methicillin resistance using cefoxitin disc (30μg). S. aureus isolates were considered methicillin-resistant if the diameter of zone of inhibition was 21mm or less (CLSI, 2017). The S. aureus isolates were then tested for mupirocin resistance. Disk diffusion method was utilized with 5μg and 200μg mupirocin discs to determine low-level and high-level resistances respectively. The criterion employed for interpretation of mupirocin resistance was a combination of the widely accepted criterion described by Finlay, Miller, and Poupard (1997) for low-level mupirocin resistance and CLSI (2017) criterion for high-level mupirocin resistance. If both inhibition zone diameters for 5μg disk and 200μg were ≥14mm, the isolate was considered mupirocin sensitive. If 5μg disc displays <14mm and 200 μg disk displayed ≥14mm inhibition zone diameter, the isolate was considered to be mupirocin low level resistant. If there is no inhibition zone in 200μg disk, the isolate was considered as mupirocin high level resistant. Results : From the 45 nasal carriage isolates, 33 (73%) were Methicillin sensitive Staphylococcus aureus (MSSA) and 12 (27%) were Methicillin Resistant Staphylococcus aureus (MRSA). Among the clinical isolates, majority (n=158, 63%) were MRSA while only 91 (37%) MSSA. An overall mupirocin resistance rate of 4.4% among S. aureus was observed. Low-level mupirocin resistance was observed in 3.7% Staphylococcus aureus isolates and high-level mupirocin resistance was observed in 0.7% isolates. Mupirocin low-level and high-level resistance in MRSA isolates were 5.3% and 0.6% respectively. MSSA isolates demonstrated 1.6% (n=2) and 0.8% (n=1) mupirocin low-level and high-level resistances respectively. None of the nasal isolates were resistant to mupirocin while 6% (n=15) mupirocin low-level resistance and 0.8% (n=2) mupirocin high-level resistance was observed in clinical isolates. Conclusion : This initial survey of mupirocin resistance among S. aureus in a country with fairly high usage of mupirocin emphasizes that although the overall mupirocin resistance is relatively low in this population, regular surveillance of mupirocin resistance remains a necessity.

Occurrence and Molecular Characterization of Abundant tet(X) Variants Among Diverse Bacterial Species of Chicken Origin in Jiangsu, China

Many novel tigecycline-inactivating enzymes encoded by tet(X) variants from different bacteria were discovered since the plasmid-mediated tet(X3) and tet(X4) genes conferring high-level resistance to tigecycline in Enterobacterales and Acinetobacter were reported. However, there have been no comprehensive studies of the prevalence of different tet(X) variants in poultry farms. In this study, we collected 45 chicken fecal samples, isolated tet(X)-positive strains, and performed antimicrobial susceptibility testing, conjugation assay, whole-genome sequencing, and bioinformatics analysis. A total of 15 tet(X)-bearing strains were isolated from 13 samples. Species identification and tet(X) subtyping analysis found that the 15 strains belonged to eight different species and harbored four different tet(X) variants. Genomic investigation showed that transmission of tet(X) variants was associated with various mobile genetic elements, and tet(X4) was the most prevalent variant transferred by conjugative plasmids. Meanwhile, we characterized a plasmid co-harboring tet(X6) and blaOXA–58 in Acinetobacter baumannii. In summary, we demonstrated that different tet(X) variants were widely disseminated in the chicken farming environment and dominated by tet(X4). This finding expands the understanding of the prevalence of tet(X) among different animal sources, and it was advocated to reduce the usage of antibiotics to limit the emergence and transmission of novel tet(X) variants in the poultry industry.

Ultrasensitive and rapid identification of ESRI developer- and piperacillin/tazobactam-resistant Escherichia coli by the MALDIpiptaz test

The excessive use of piperacillin/tazobactam (P/T) has promoted the emergence of P/T-resistant Enterobacterales. We reported that in Escherichia coli, P/T contributes to the development of extended-spectrum resistance to β-lactam/β-lactamase inhibitor (BL/BLI) (ESRI) in isolates that are P/T susceptible but have low-level resistance to BL/BLI. Currently, the detection of P/T resistance relying on conventional methods is time-consuming. To overcome this issue, we developed a cost-effective test based on MALDI-MS technology, called MALDIpiptaz, which aims to detect P/T resistance and ESRI developers in E. coli. We used automated Clover MS Data Analysis software to analyse the protein profile spectra obtained by MALDI-MS from a collection of 248 E. coli isolates (91 P/T-resistant, 81 ESRI developers and 76 P/T-susceptible). This software allowed to preprocess all the spectra to build different peak matrices that were analysed by machine learning algorithms. We demonstrated that MALDIpiptaz can efficiently and rapidly (15 min) discriminate between P/T-resistant, ESRI developer and P/T-susceptible isolates and allowed the correct classification between ESRI developers from their isogenic resistance to P/T. The combination of excellent performance and cost-effectiveness are all desirable attributes, allowing the MALDIpiptaz test to be a useful tool for the rapid determination of P/T resistance in clinically relevant gram-negative bacteria.

The Prevalence and Determinants of Fusidic Acid Resistance Among Methicillin-Resistant Staphylococcus aureus Clinical Isolates in China

The significant increase in resistance of methicillin-resistant Staphylococcus aureus (MRSA) to fusidic acid (FA) is a worrying public concern. However, the data on the prevalence of FA-resistant MRSA isolates in China is still limited. This study aims to investigate the prevalence of FA resistance and resistance determinants among MRSA isolates from six tertiary hospitals in different regions of China between 2016 and 2020. The antimicrobial susceptibility of MRSA isolates was performed by disk diffusion test and broth microdilution method. Whole-genome sequencing was conducted to evaluate the determinants of FA resistance and molecular characterization of FA-resistant MRSA isolates. In this study, a total of 74 (74/457, 16.2%) isolates were identified to be FA-resistant among 457 non-duplicate MRSA isolates. The prevalence of 74 FA-resistant isolates was as follows: Hubei (28/70, 40%), Shanghai (18/84, 21.4%), Jiangxi (7/58, 12.1%), Inner Mongolia Autonomous Region (6/38, 15.8%), Guangdong (12/112, 10.7%), and Sichuan (3/95, 3.2%). The mutations in fusA were present in 79.7% (59/74) of FA-resistant MRSA isolates, with 54 (54/74, 73%) having L461K mutation and conferring high-level resistance [Minimum Inhibitory Concentration (MIC)&gt;128 μg/ml]. Acquired gene, fusB, with low-level resistance (MIC &lt;16 μg/ml) was found in 20.3% (15/74) FA-resistant MRSA isolates. ST5-MRSA-II-t2460 was the most prevalence clone with high-level resistance, accounting for 51.4% (38/74), which was distributed in Hubei (24/28, 85.7%), Inner Mongolia Autonomous Region (4/6, 66.7%), Shanghai (7/18, 38.9%), and Guangdong (3/12, 25%). ST630-t4549 MRSA isolates with low-level resistance were the most common in Jiangxi (3/7, 42.9%) and Sichuan (2/3, 66.7%). In brief, the prevalence of FA resistance among MRSA isolates in China was relatively high with geographic differences. High-level FA resistance was associated mostly with fusA mutations, especially the L461K mutation, whereas fusB usually conferred the low-level resistance to FA. The spread of ST5-MRSA-II-t2460 clone with high-level resistance to FA contributed greatly to the increase of FA-resistant MRSA isolates in most regions, especially in Hubei.

Whole-Genome Sequencing to Identify Missed Rifampicin and Isoniazid Resistance Among Tuberculosis Isolates—Chennai, India, 2013–2016

India has a high burden of drug-resistant tuberculosis (DR TB) and many cases go undetected by current drug susceptibility tests (DSTs). This study was conducted to identify rifampicin (RIF) and isoniazid (INH) resistance associated genetic mutations undetected by current clinical diagnostics amongst persons with DR TB in Chennai, India. Retrospectively stored 166 DR TB isolates during 2013–2016 were retrieved and cultured in Löwenstein-Jensen medium. Whole genome sequencing (WGS) and MGIT DST for RIF and INH were performed. Discordant genotypic and phenotypic sensitivity results were repeated for confirmation and the discrepant results considered final. Further, drug resistance-conferring mutations identified through WGS were analyzed for their presence as targets in current WHO-recommended molecular diagnostics. WGS detected additional mutations for rifampicin and isoniazid resistance than WHO-endorsed line probe assays. For RIF, WGS was able to identify an additional 10% (15/146) of rpoB mutant isolates associated with borderline rifampicin resistance compared to MGIT DST. WGS could detect additional DR TB cases than commercially available and WHO-endorsed molecular DST tests. WGS results reiterate the importance of the recent WHO revised critical concentrations of current MGIT DST to detect low-level resistance to rifampicin. WGS may help inform effective treatment selection for persons at risk of, or diagnosed with, DR TB.

Identification of Multiple Low-Level Resistance Determinants and Coselection of Motility Impairment upon Sub-MIC Ceftriaxone Exposure in Escherichia coli

Ceftriaxone is a widely consumed antibiotic used to treat bacterial infections. Bacteria, however, are increasingly becoming resistant to ceftriaxone.