BACKGROUND: Typhoid fever, caused by Salmonella enterica serovar Typhi, is a significant public health concern due to the escalating of antimicrobial resistance (AMR), with limited treatment options for extensively drug-resistant (XDR) S. Typhi strains pose a serious threat to disease management and control. This study aimed to investigate the genomic characteristics, epidemiology and AMR genes of XDR S. Typhi strains from typhoid fever patients in Pakistan.
METHODS: We assessed 200 patients with enteric fever symptoms, confirming 65 S. Typhi cases through culturing and biochemical tests. Subsequent antimicrobial susceptibility testing revealed 40 cases of extensively drug-resistant (XDR) and 25 cases of multi-drug resistance (MDR). Thirteen XDR strains were selected for whole-genome sequencing, to analyze their sequence type, phylogenetics, resistance genes, pathogenicity islands, and plasmid sequences using variety of data analysis resources. Pangenome analysis was conducted for 140 XDR strains, including thirteen in-house and 127 strains reported from other regions of Pakistan, to assess their genetic diversity and functional annotation.
RESULTS: MLST analysis classified all isolates as sequence type 1 (ST-1) with 4.3.1.1. P1 genotype characterization. Prophage and Salmonella Pathogenicity Island (SPI) analysis identified intact prophages and eight SPIs involved in Salmonella\'s invasion and replication within host cells. Genome data analysis revealed numerous AMR genes including dfrA7, sul1, qnrS1, TEM-1, Cat1, and CTX-M-15, and SNPs associated with antibiotics resistance. IncY, IncQ1, pMAC, and pAbTS2 plasmids, conferring antimicrobial resistance, were detected in a few XDR S. Typhi strains. Phylogenetic analysis inferred a close epidemiological linkage among XDR strains from different regions of Pakistan. Pangenome was noted closed among these strains and functional annotation highlighted genes related to metabolism and pathogenesis.
CONCLUSIONS: This study revealed a uniform genotypic background among XDR S. Typhi strains in Pakistan, signifying a persistence transmission of a single, highly antibiotic-resistant clone. The closed pan-genome observed underscores limited genetic diversity and highlights the importance of genomic surveillance for combating drug-resistant typhoid infections.

An extensively-drug resistant (XDR) Escherichia coli W60 was isolated from the urine sample of a patient. The genetic basis for its XDR phenotype was investigated, particularly the basis for its resistance toward β-lactam/BLI (β-Lactamase Inhibitor) combinations. Following determination of the XDR phenotype, third generation genomic sequencing was performed to identify genetic structures in E. coli W60. Further cloning analysis was performed to identify determinants of β-lactam/BLI combination resistance. It was found that E. coli W60 is resistant to nearly all of the tested antibiotics including all commonly used β-lactam/BLI combinations. Analysis of the genomic structures in E. coli W60 showed two novel transferable plasmids are responsible for the resistance phenotypes. Further genetic analysis showed bla NDM-5 leads to high resistance to β-lactam/BLI combinations, which was enhanced by co-expressing ble MBL. pECW602 harbors a truncated bla TEM that is not functional due to the loss of the N-terminal signal peptide coding region. Research performed in this work leads to several significant conclusions: the XDR phenotype of E. coli W60 can be attributed to the presence of transferable multidrug resistance plasmids; NDM-5 confers high resistance to β-lactam/BLI combinations; co-expression of ble MBL enhances resistance caused by NDM-5; the signal peptides of TEM type β-lactamases are essential for their secretion and function. Findings of this work show the danger of transferable multidrug resistance plasmids and metallo-β-lactamases, both of which should be given more attention in the analysis and treatment of multidrug resistant pathogens.

Multi-, extensively-, and pan-drug resistant bacteria are a threat to our health today, because their wide resistance spectra make their infections difficult to cure. In this work, we isolated an extensively drug resistant (XDR) Klebsiella pneumoniae 2-1 strain from the stool sample of a patient diagnosed of colorectal cancer. K. pneumoniae 2-1 was found to be resistant to all the antibiotics tested except for cefepime, tigecycline, and ceftazidime-avibactam. By sequencing the complete genome of K. pneumoniae 2-1, we found it contains a chromosome of 5.23 Mb and two circular plasmids with the size of 246 and 90 kb. The larger plasmid, pKP21HI1 was found to be a new conjugation-defective plasmid belonging to incompatibility group HI1B and a new sequence type. Further comparative genomics analysis and antimicrobial resistance gene analysis showed that although a great deal of changes took place on the chromosome of K. pneumoniae 2-1 in comparison with the reference genome, the extensively drug resistance phenotype of K. pneumoniae 2-1 is primarily due to the two multidrug resistant plasmids it contains. This work explains the genetic and mechanistic basis of the extensive drug resistance of K. pneumoniae 2-1, and found that plasmids play key roles in the strong antibiotic resistance of bacteria.

The implementation of rapid and reliable drug susceptibilities diagnosis is fundamental for effective treatment of multidrug-resistant tuberculosis(MDR-TB). The present study aimed to assess the diagnostic performance of the 2nd-version GenoType MTBDRsl kit as well as the impact of its implementation on the turnaround time in a multi-center Chinese study.
Totally 353 MDR-TB patient specimens were consecutively tested. The 2nd-version GenoType MTBDRsl assay, drug susceptibility testing with the MGIT 960 system, and sequencing were performed and compared.
MTBDRsl testing identified the major genotypes associated with fluoroquinolones resistance, predominated by gyrA MUT3B (Asp94Asn and Asp94Tyr, 26.5%) and MUT3C (Asp94Gly, 19.5%). The genotypes associated with resistance to 2nd-line injectable drugs(SLIDs) were rrsMUT1(A1401G, 64.9%) and absence of WT1(C1402T, 10.5%). The sensitivities for detection of resistance to fluoroquinolones, SLIDs, and their combination (extensively drug resistance, XDR) were 80.5%, 80.7% and 73.5% and specificities were 100.0%, 99.3% and 99.1%, respectively. Implementation of this test significantly reduced the turnaround time between sample collection and result reporting from 45 to 3 days, a reduction by 93.3% (p, 0.001).
With a favorable diagnostic performance and short turnaround time, the 2nd-version GenoType MTBDRsl assay proves its value for early diagnosis of resistance to 2nd-line drugs as well as of XDR-TB in China.

The prevalence of extensively drug-resistant (XDR) and pre-extensively drug-resistant (pre-XDR) tuberculosis in China highlights the need for rapid diagnosis. Molecular methods based on the detection of resistance-conferring mutations provide promising solution for rapid diagnosis. Here, we evaluated the accuracy of using mutations in gyrA, rrs and tlyA to predict resistance to levofloxacin (LEV), amikacin (AMK) and capreomycin (CAP), among 208 clinical multidrug-resistant (MDR) Mycobacterium tuberculosis strains collected in a local hospital in Shandong province, China. A total of 131 (63.0%, 131/208) strains were detected resistance to at least one of the 3-s line drugs by drug susceptible tests (DSTs). By comparing the mutation data with the phenotypic results, we found all mutations in three genes could specifically (with specificities from 93.9% to 100%) predict resistances with sensitivities of 77.8% for gyrA (LEV), 71.4% for rrs (AMK), 53.6% for rrs (CAP), 14.3% for tlyA (CAP), 64.3% for rrs and tlyA (CAP). The combination of these mutations could predict 68.9% and 63.4% pre-XDR and XDR TB, respectively. However, the positive predictive value of rrs for CAP resistance (57.7%) and the negative predictive values of gyrA for LEV resistance (74.5%) were not satisfying. Our results supported the use of genetic mutations to predict resistance to AMK and fluoroquinolones. An algorithm that combines molecular methods and traditional DST would be valuable for detecting resistance to second-line drugs in our hospital.