Metabolic remodeling by RNA polymerase gene mutations is associated with reduced ß-lactam susceptibility in oxacillin-susceptible MRSA.

The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to ß-lactam antibiotics, these strains can easily acquire reduced ß-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced ß-lactam susceptibility, as it does not directly associate with the expression of mecA. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoBH929P, RpoBQ645H, RpoCG950R, RpoCG498D, RpiAA64E, and FruBA211E, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. IMPORTANCE: The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to ß-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC, leading to treatment failure. This study investigated the mechanisms underlying reduced ß-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

A multidrug-resistant Stenotrophomonas maltophilia clinical isolate from Kamuzu Central Hospital, Malawi.

Background: Stenotrophomonas maltophilia is a significant opportunistic pathogen that is associated with high mortality in immunocompromised individuals. In this study, we describe a multidrug-resistant (MDR) S. maltophilia clinical isolate from Kamuzu Central Hospital (KCH), Lilongwe, Malawi. Methods: A ceftriaxone and meropenem nonsusceptible isolate (Sm-MW08), recovered in December 2017 at KCH, was referred to the National Microbiology Reference Laboratory for identification. In April 2018, we identified the isolate using MALDI Biotyper mass spectrometry and determined its antimicrobial susceptibility profile using microdilution methods. Sm-MW08 was analysed by S1-PFGE, PCR, and Sanger sequencing, in order to ascertain the genotypes that were responsible for the isolate's multidrug-resistance (MDR) phenotype. Results: Sm-MW08 was identified as S. maltophilia and exhibited resistance to a range of antibiotics, including all ß-lactams, aminoglycosides (except arbekacin), chloramphenicol, minocycline, fosfomycin and fluoroquinolones, but remained susceptible to colistin and trimethoprim-sulfamethoxazole. The isolate did not harbour any plasmid but did carry chromosomally-encoded blaL1 metallo-ß-lactamase and blaL2 ß-lactamase genes; this was consistent with the isolate's resistance profile. No other resistance determinants were detected, suggesting that the MDR phenotype exhibited by Sm-MW08 was innate. Conclusion: Herein, we have described an MDR S. maltophilia from KCH in Malawi, that was resistant to almost all locally available antibiotics. We therefore recommend the practice of effective infection prevention measures to curtail spread of this organism.

A multidrug-resistant Stenotrophomonas maltophilia clinical isolate from Kamuzu Central Hospital, Malawi

Background Stenotrophomonas maltophilia is a significant opportunistic pathogen that is associated with high mortality in immunocompromised individuals. In this study, we describe a multidrug-resistant (MDR) S. maltophilia clinical isolate from Kamuzu Central Hospital (KCH), Lilongwe, Malawi. Methods: A ceftriaxone and meropenem nonsusceptible isolate (Sm-MW08), recovered in December 2017 at KCH, was referred to theNational Microbiology Reference Laboratory for identification. In April 2018, we identified the isolate using MALDI Biotyper mass spectrometry and determined its antimicrobial susceptibility profile using microdilution methods. Sm-MW08 was analysed by S1-PFGE, PCR, and Sanger sequencing, in order to ascertain the genotypes that were responsible for the isolate`s multidrug-resistance (MDR) phenotype. Results Sm-MW08 was identified as S. maltophilia and exhibited resistance to a range of antibiotics, including all ß-lactams, aminoglycosides (except arbekacin), chloramphenicol, minocycline, fosfomycin and fluoroquinolones, but remained susceptible to colistin and trimethoprim-sulfamethoxazole. The isolate did not harbour any plasmid but did carry chromosomally-encoded blaL1 metallo-ßlactamase and blaL2 ß-lactamase genes; this was consistent with the isolate's resistance profile. No other resistance determinants were detected, suggesting that the MDR phenotype exhibited by Sm-MW08 was innate. Conclusion : Herein, we have described an MDR S. maltophilia from KCH in Malawi, that was resistant to almost all locally available antibiotics. We therefore recommend the practice of effective infection prevention measures to curtail spread of this organism

Genomic characterisation of a novel plasmid carrying blaIMP-6 of carbapenem-resistant Klebsiella pneumoniae isolated in Osaka, Japan.

OBJECTIVES: To analyse plasmids carrying blaIMP-6 in Klebsiella pneumoniae isolates obtained from multicentre carbapenem-resistant Enterobacteriaceae surveillance. METHODS: Plasmids harbouring blaIMP-6 were characterised by the whole-genome sequencing of four Klebsiella pneumoniae isolates carrying blaIMP-6, and compared with the pKPI-6 plasmid, which is widespread in western Japan, through pulsed-field gel electrophoresis, Southern blotting, bacterial conjugation, and qPCR. RESULTS: Whole-genome sequencing analysis revealed that three of the four isolates carried approximately 50 kbp plasmids similar to the pKPI-6 plasmid; however, one isolate carried a 250 kbp plasmid harbouring blaIMP-6 (pE196_IMP6). So far, all of the reported plasmids carrying blaIMP-6 were similar to the pKPI-6 plasmid, and this plasmid was a novel blaIMP6-carrier. The size and transferability of this plasmid was confirmed by Southern hybridisation and conjugation experiments. It was demonstrated that the generation of plasmid pE196_IMP6 was due to an intramolecular transposition mediated by IS26, and a homologous recombination between plasmids pKPI-6 and pE013 that was obtained from another carbapenem-resistant Enterobacteriaceae isolate in this analysis. As a result of co-integration with pE013, pE196_IMP6 acquired six additional pairs of type II toxin-antitoxin systems that pKPI-6 does not carry. Transcription of all of the toxin-antitoxin systems were confirmed in an isolate carrying pE196_IMP6 by qPCR. CONCLUSIONS: This study detected a novel plasmid carrying blaIMP-6, and revealed the origin of this plasmid. Toxin-antitoxin system acquisition could enable pE196_IMP6 maintenance persistently through successions, even without selection pressure by the clinical usage of antimicrobials, generating broad dissemination and longer carbapenem-resistant Enterobacteriaceae colonisation duration in patients.

First Identification and genomic characterization of multidrug-resistant carbapenemase-producing Enterobacteriaceae clinical isolates in Malawi, Africa.

Purpose. Carbapenemase-producing Enterobacteriaceae (CPE) have become a global concern and a serious threat to human health due to their resistance to multiple antibiotics. In this study, we identified and characterized CPE for the first time in Malawi, southeastern Africa.Methodology. We investigated the possible presence of carbapenemases among a collection of 200 ceftriaxone-nonsusceptible Gram-negative clinical isolates obtained from five Malawian hospitals between January 2016 and December 2017, using both phenotypic and genotypic tests. Molecular typing of CPE was done by PFGE, multilocus sequence typing (ST) or phylogenetic grouping. Resistant plasmids were characterized by S1 PFGE, Southern blotting and conjugation assays.Results. Out of 200 isolates, we detected 16 (8 %) CPE of which all originated from one referral hospital, Kamuzu Central Hospital, in the Central part of Malawi. Of 16 isolates, seven Klebsiella pneumoniae ST340/CC258 carried bla KPC-2, two Escherichia coli ST636 (phylogroup B2) carried bla NDM-5, six E. coli ST617 (phylogroup A) and one Klebsiella variicola carried bla OXA-48. All carbapenemases were plasmid-encoded, but only bla NDM-5-carrying plasmids could be conjugated. Most isolates co-harboured other ß-lactamases and consequently exhibited a wider spectrum of resistance to commonly used antibiotics. We observed indistinguishable genetic profiles between strain types, despite originating from different wards, suggesting acquisition during admission and intra-hospital spread.Conclusion. This report strongly suggests a probable existence of highly resistant various types of CPE organisms in Malawi including KPC-2-producing K. pneumoniae ST340/CC258, a known high-risk epidemic lineage.

The occurrence and frequency of genomic mutations that mediate Isoniazid and Rifampicin resistance in Mycobacterium tuberculosis isolates from untreated pulmonary Tuberculosis cases in urban Blantyre, Malawi.

Background: The emergence and spread of drug-resistant Tuberculosis (TB) is a major public health threat. TB resistance originates in the course of treatment due to genomic mutations in Mycobacterium tuberculosis (MTB). An increase in new cases with drug-resistant TB could be an indicator of high levels of circulating resistant strains. This study was conducted to determine the occurrence and frequency of genomic mutations that mediate Isoniazid (INH) and Rifampicin (RIF) resistance among isolates from untreated TB cases in urban Blantyre, Malawi. Methods: A cross-sectional retrospective study was conducted on a panel of 141(n=141) MTB clinical isolates recovered between June 2010 and January 2012 from >2+ Ziehl-Neelsen smear positive new pulmonary-TB patients with no history of treatment. Frozen isolates were revived using the BACTEC MGIT detection system. DNA was extracted using GenoLyse DNA extraction kit and detection of genomic mutations was carried out using the GenoType MTBDRplus Ver 2.0 assay. Results: Out of the 141 isolates studied, 3 (2.1%) were found carrying mutations in the katG gene that confer resistance to Isoniazid (INH). No mutations were detected in the inhA promoter region gene that confer weak INH resistance or in the rpoB gene that confer Rifampicin resistance. All katG mutant genes had a S315T1 single point mutation, a genomic alteration that mediates high INH resistance. Conclusion: The katG mutant gene conferring resistance to INH was the only genomic mutation observed among the isolates studied and the frequency of occurrence was low. Our findings suggest low levels of circulating drug-resistant MTB strains in urban Blantyre, Malawi.