Whole genome sequencing reveals high-resolution epidemiological links between clinical and environmental Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a gram-negative bacterial species capable of occupying a broad range of environmental and clinical habitats. Known as an opportunistic pathogen, it has recently become a major causative agent of clinical infections worldwide. Despite growing knowledge about the highly diverse population of K. pneumoniae, the evolution and clinical significance of environmental K. pneumoniae, as well as the relationship between clinical and environmental K. pneumoniae, are poorly defined. METHODS: We isolated and sequenced K. pneumoniae from in-patients in a single hospital in Thailand, as well as hospital sewage, and surrounding canals and farms within a 20-km radius. RESULTS: Phylogenetic analysis of 77 K. pneumoniae (48 clinical and 29 non-clinical isolates) demonstrated that the two groups were intermixed throughout the tree and in some cases resided in the same clade, suggesting recent divergence from a common ancestor. Phylogenetic comparison of the 77 Thai genomes with 286 K. pneumoniae from a global collection showed that Thai isolates were closely related to the clinical sub-population of the global collection, indicating that Thai clinical isolates belonged to globally circulating lineages. Dating of four Thai K. pneumoniae clades indicated that they emerged between 50 and 150 years ago. Despite their phylogenetic relatedness, virulence factors and β-lactamase resistance genes were more numerous in clinical than in environmental isolates. Our results indicate that clinical and environmental K. pneumoniae are closely related, but that hospitals may select for isolates with a more resistant and virulent genotype. CONCLUSIONS: These findings highlight the clinical relevance of environmental K. pneumoniae isolates

Whole genome sequencing of ESBL-producing Escherichia coli isolated from patients, farm waste and canals in Thailand.

BACKGROUND: Tackling multidrug-resistant Escherichia coli requires evidence from One Health studies that capture numerous potential reservoirs in circumscribed geographic areas. METHODS: We conducted a survey of extended β-lactamase (ESBL)-producing E. coli isolated from patients, canals and livestock wastewater in eastern Thailand between 2014 and 2015, and analyzed isolates using whole genome sequencing. RESULTS: The bacterial collection of 149 isolates consisted of 84 isolates from a single hospital and 65 from the hospital sewer, canals and farm wastewater within a 20 km radius. E. coli ST131 predominated the clinical collection (28.6%), but was uncommon in the environment. Genome-based comparison of E. coli from infected patients and their immediate environment indicated low genetic similarity overall between the two, although three clinical-environmental isolate pairs differed by ≤ 5 single nucleotide polymorphisms. Thai E. coli isolates were dispersed throughout a phylogenetic tree containing a global E. coli collection. All Thai ESBL-positive E. coli isolates were multidrug resistant, including high rates of resistance to tobramycin (77.2%), gentamicin (77.2%), ciprofloxacin (67.8%) and trimethoprim (68.5%). ESBL was encoded by six different CTX-M elements and SHV-12. Three isolates from clinical samples (n = 2) or a hospital sewer (n = 1) were resistant to the carbapenem drugs (encoded by NDM-1, NDM-5 or GES-5), and three isolates (clinical (n = 1) and canal water (n = 2)) were resistant to colistin (encoded by mcr-1); no isolates were resistant to both carbapenems and colistin. CONCLUSIONS: Tackling ESBL-producing E. coli in this setting will be challenging based on widespread distribution, but the low prevalence of resistance to carbapenems and colistin suggests that efforts are now required to prevent these from becoming ubiquitous

Host genetic polymorphisms involved in long-term symptoms of COVID-19

ABSTRACTHost genetic polymorphisms are recognized as a critical determinant of diversity in clinical symptoms of Coronavirus disease 2019 (COVID-19). Accordingly, this study aimed to determine possible associations between single nucleotide polymorphisms (SNPs) in 37 candidate genetic variants and clinical consequences of COVID-19 – especially long-term symptoms, Long COVID. A total of 260 COVID-19 patients, divided into mild (n = 239) and severe (n = 21) and further categorized based on the presence of Long COVID (no, n = 211; yes, n = 49), were recruited. Genotyping of selected polymorphisms responsible for viral entry, immune response, and inflammation was performed using MassARRAY system. Out of 37 SNPs, 9 including leucine zipper transcription factor like-1 (LZTFL1) rs10490770 C allele, LZTFL1 rs11385942 dupA allele, nicotinamide adenine dinucleotide synthetase-1 (NADSYN1) rs12785878 TT genotype, plexin A-4 (PLXNA4) rs1424597 AA genotype, LZTFL1 rs17713054 A allele, interleukin-10 (IL10) rs1800896 TC genotype and C allele, angiotensin converting enzyme-2 (ACE2) rs2285666 T allele, and plasmanylethanolamine desaturase-1 (PEDS1) rs6020298 GG genotype and G allele were significantly associated with an increased risk of developing Long COVID, whereas interleukin-10 receptor subunit beta (IL10RB) rs8178562 GG genotype was significantly associated with a reduced risk of Long COVID. Kaplan-Meier curve displayed that the above gene polymorphisms were significantly associated with cumulative rate of Long COVID occurrence. Polymorphisms in LZTFL1 rs10490770, LZTFL1 rs11385942, LZTFL1 rs17713054, NADSYN1 rs12785878, PLXNA4 rs1424597, IL10 rs1800896, ACE2 rs2285666, PEDS1 rs6020298, and IL10RB rs8178562 appear to be genetic factors involved in development of Long COVID

Miniaturised broth microdilution for simplified antibiotic susceptibility testing of Gram negative clinical isolates using microcapillary devices

Antibiotic resistance is a major global challenge. Although microfluidic antibiotic susceptibility tests (AST) offer great potential for rapid and portable testing to inform correct antibiotic selection, the impact of miniaturisation on broth microdilution (BMD) is not fully understood. We developed a 10-plex microcapillary based broth microdilution using resazurin as a colorimetric indicator for bacterial growth. Each capillary had a 1 microlitre capillary volume, 100 times smaller than microplate broth microdilution. The microcapillary BMD was compared to an in-house standard microplate AST and commercial Vitek 2 system. When tested with 25 uropathogenic isolates (20 Escherichia coli and 5 Klebsiella pneumoniae) and 2 reference E. coli, these devices gave 96.1% (441/459 isolate/antibiotic combinations) categorical agreement, across 17 therapeutically beneficial antibiotics, compared to in-house microplate BMD with resazurin. A further 99 (50 E. coli and 49 K. pneumoniae) clinical isolates were tested against 10 antibiotics and showed 92.3% categorical (914/990 isolate/antibiotic combinations) compared to the Vitek 2 measurements. These microcapillary tests showed excellent analytical agreement to existing AST methods. Furthermore, the small size and simple colour change can be recorded using a smartphone camera or it is feasible to follow growth kinetics using very simple, low-cost readers. The test strips used here are produced in large batches, allowing hundreds of multiplex tests to be made and tested rapidly. Demonstrating performance of miniaturised broth microdilution with clinical isolates paves the way for wider use of microfluidic AST

Additional file 4: Table S3. of Whole genome sequencing reveals high-resolution epidemiological links between clinical and environmental Klebsiella pneumoniae

Supplemental Table S3, which includes the list of β-lactamase genes and ESBLs. (CSV 5 kb

Additional file 2: Figures S1–S6. of Whole genome sequencing reveals high-resolution epidemiological links between clinical and environmental Klebsiella pneumoniae

Figures and descriptions of Supplemental Figures S1–S6 mentioned throughout the manuscript. (DOCX 1927 kb

Additional file 3: Table S2. of Whole genome sequencing reveals high-resolution epidemiological links between clinical and environmental Klebsiella pneumoniae

Supplemental Table S2, which includes the list of putative virulence factor genes. (CSV 9 kb

Additional file 2: Table S2. of Whole genome sequencing of ESBL-producing Escherichia coli isolated from patients, farm waste and canals in Thailand

Sequence quality metrics. (XLSX 495 kb

Additional file 1: Table S1. of Whole genome sequencing of ESBL-producing Escherichia coli isolated from patients, farm waste and canals in Thailand

Isolate details. (XLSX 15 kb

Additional file 4: Table S3. of Whole genome sequencing of ESBL-producing Escherichia coli isolated from patients, farm waste and canals in Thailand

Genetic context of mcr-1 and carbapenem resistance genes. (XLSX 9 kb