Genomic diversity and antimicrobial resistance in clinical Klebsiella pneumoniae isolates from tertiary hospitals in Southern Ghana.

OBJECTIVES: Comprehensive data on the genomic epidemiology of hospital-associated Klebsiella pneumoniae in Ghana are scarce. This study investigated the genomic diversity, antimicrobial resistance patterns, and clonal relationships of 103 clinical K. pneumoniae isolates from five tertiary hospitals in Southern Ghana-predominantly from paediatric patients aged under 5 years (67/103; 65%), with the majority collected from urine (32/103; 31%) and blood (25/103; 24%) cultures. METHODS: We generated hybrid Nanopore-Illumina assemblies and employed Pathogenwatch for genotyping via Kaptive [capsular (K) locus and lipopolysaccharide (O) antigens] and Kleborate (antimicrobial resistance and hypervirulence) and determined clonal relationships using core-genome MLST (cgMLST). RESULTS: Of 44 distinct STs detected, ST133 was the most common, comprising 23% of isolates (n = 23/103). KL116 (28/103; 27%) and O1 (66/103; 64%) were the most prevalent K-locus and O-antigen types. Single-linkage clustering highlighted the global spread of MDR clones such as ST15, ST307, ST17, ST11, ST101 and ST48, with minimal allele differences (1-5) from publicly available genomes worldwide. Conversely, 17 isolates constituted novel clonal groups and lacked close relatives among publicly available genomes, displaying unique genetic diversity within our study population. A significant proportion of isolates (88/103; 85%) carried resistance genes for ≥3 antibiotic classes, with the blaCTX-M-15 gene present in 78% (n = 80/103). Carbapenem resistance, predominantly due to blaOXA-181 and blaNDM-1 genes, was found in 10% (n = 10/103) of the isolates. CONCLUSIONS: Our findings reveal a complex genomic landscape of K. pneumoniae in Southern Ghana, underscoring the critical need for ongoing genomic surveillance to manage the substantial burden of antimicrobial resistance.

The dCas9-based genome editing in Plasmodium yoelii.

Genetic editing is a powerful tool for functional characterization of genes in various organisms. With its simplicity and specificity, the CRISPR-Cas9 technology has become a popular editing tool, which introduces site-specific DNA double-strand breaks (DSBs), and then leverages the endogenous repair pathway for DSB repair via homology-directed repair (HDR) or the more error-prone non-homologous end joining (NHEJ) pathways. However, in the Plasmodium parasites, the lack of a typical NHEJ pathway selects for DSB repair through the HDR pathway when a homologous DNA template is available. The AT-rich nature of the Plasmodium genome exacerbates this drawback by making it difficult to clone longer homologous repair DNA templates. To circumvent these challenges, we adopted the hybrid catalytically inactive Cas9 (dCas9)-microbial single-stranded annealing proteins (SSAP) editor to the Plasmodium genome. In Plasmodium yoelii, we demonstrated the use of the dCas9-SSAP, as the cleavage-free gene editor, by targeted gene deletion and gene tagging, even using shorter homologous DNA templates. This dCas9-SSAP method with a shorter DNA template, which did not require DSBs, independent of HDR and NHEJ, would be a great addition to the existing genetic toolbox and could be deployed for the functional characterization of genes in Plasmodium, contributing to improving the ability of the malaria research community in characterizing more than half of genes with unknown functions.IMPORTANCEMalaria caused by Plasmodium parasites infection remains a serious threat to human health, with an estimated 249 million malaria cases and 608,000 deaths worldwide in 2022, according to the latest report from the World Health Organization (WHO). Here, we demonstrated the use of dCas9-single-stranded annealing protein, as the cleavage-free gene editor in Plasmodium yoelii, by targeted deletion and gene tagging, even using shorter homologous DNA templates. This method with a shorter DNA template, which did not require DSBs, independent of HDR and NHEJ, showing the potential significance in greatly improving our ability to elucidate gene functions, would contribute to assisting the malaria research community in deciphering more than half of genes with unknown functions to identify new drug and vaccine targets.

CRISPR-Cas13 in malaria parasite: Diagnosis and prospective gene function identification.

Malaria caused by Plasmodium is still a serious public health problem. Genomic editing is essential to understand parasite biology, elucidate mechanical pathways, uncover gene functions, identify novel therapeutic targets, and develop clinical diagnostic tools. Recent advances have seen the development of genomic diagnostic technologies and the emergence of genetic manipulation toolbox comprising a host of several systems for editing the genome of Plasmodium at the DNA, RNA, and protein level. Genomic manipulation at the RNA level is critical as it allows for the functional characterization of several transcripts. Of notice, some developed artificial RNA genome editing tools hinge on the endogenous RNA interference system of Plasmodium. However, Plasmodium lacks a robust RNAi machinery, hampering the progress of these editing tools. CRISPR-Cas13, which belongs to the VI type of the CRISPR system, can specifically bind and cut RNA under the guidance of crRNA, with no or minimal permanent genetic scar on genes. This review summarizes CRISPR-Cas13 system from its discovery, classification, principle of action, and diagnostic platforms. Further, it discusses the application prospects of Cas13-based systems in Plasmodium and highlights its advantages and drawbacks.

Resistance Status of Bacteria from a Health Facility in Ghana: A Retrospective Study.

BACKGROUND: Regardless of the global concerted effort to control the development and spread of antimicrobial resistance, increasing cases are continually documented at many medical centres. This situation is reinforced by inadequate information on the trend of resistance resulting from lack of regular antimicrobial resistance surveillance. The present study sought to detect the number of multidrug-resistant (MDR), extended drug-resistant (XDR), and pandrug-resistant (PDR) bacterial isolates at a health facility in Ghana from January 2018 to July 2020. METHOD: A total of 800 data on antimicrobial testing results were extracted from the records of the health facility. The extracted data were explored for the detection of MDR, XDR, and PDR. The study further determined the use of antibiotics using the multiple-drug resistance index (MDRI). RESULTS: Except for Staphylococcus and Neisseria spp., all bacterial isolates showed extremely high (100%) proportion of MDR. Although only Staphylococcus spp. (38 (4.8%)) was observed to be XDR, the rest of the bacteria showed the potential to attain the status of XDR or PDR. MDRI indicated high use of antibiotics in the health facility. CONCLUSION: The high antimicrobial resistance observed by the study underscores the need for prompt and effective antibiotic resistance control strategies.

The Effectiveness of Dipstick for the Detection of Urinary Tract Infection.

BACKGROUND: The balance between the choices of UTI diagnostic tools in most primary care settings has been settled for by the more rapid, less labour-intensive dipstick. This study aimed to evaluate the effectiveness of dipstick for diagnosing UTI. METHOD: A total of 429 urine samples were collected from patients suspected of UTI; cultured on cysteine-lactose-electrolyte-deficient (CLED) agar, blood agar, and MacConkey agar; and incubated at 37°C overnight. Urine cultures with bacteria count ≥105 cfu/ml were classified as "positive" for UTI. A dipstick was used to screen for the production of nitrite (NIT) and leucocyte esterase (LE), following the manufacturer's instructions. Biochemical reactions of nitrite and leucocyte esterase > "trace" were classified as "positive." A quantitative urine culture was used as the gold standard. RESULTS: The highest sensitivity value and negative predictive value were recorded for the combined "NIT+ or LE+" dipstick results. The highest specificity value, positive predictive value, positive likelihood ratio, and negative likelihood ratio were recorded for "nitrite-positive and leucocyte esterase-positive" results. Combined "nitrite-positive or leucocyte-positive" result was relatively the best indicator for accurate dipstick diagnosis, with AUC = 0.7242. Cohen's kappa values between dipstick diagnosis and quantitative culture were <0.6. CONCLUSION: Combined performance of nitrite and leucocyte esterase results appeared better than the solo performance of nitrite and leucocyte esterase. However, little confidence should be placed on dipstick diagnosis; hence, request for quantity culture should be encouraged in the primary healthcare settings.

Geographical Distribution of ß-Lactam Resistance among Klebsiella spp. from Selected Health Facilities in Ghana.

ß-Lactam-resistant Klebsiella isolates continue to cause multidrug resistance infections worldwide. This study aimed to describe the geographical distribution of extended spectrum ß-lactamase (ESBL), AmpC ß-lactamase (AmpC), and carbapenemase production among 139 Klebsiella isolates recovered from patients at major referral health facilities in Ghana. The phenotypic methods of combined disc diffusion test, modified three-dimensional test, modified Hodge test (MHT), and combined disc test were performed for each isolate to detect ESBL, AmpC, carbapenemase, and metallo-ß-lactamase (MBL) producers, respectively. Except for MBL, all other ß-lactam resistance mechanisms were highest in the healthcare facilities situated in the northern belt of Ghana. Significant regional difference of ESBL producers was observed between the northern and middle belts as well as the northern and southern belts. Genotypic detection with polymerase chain reaction (PCR) revealed the presence of bla TEM 36/139 (25.9%), bla SHV 40/139 (28.8%), bla CTX-M 37/139 (26.6%), bla OXA-48 3/139 (2.16%), and bla NDM 1/139 (0.72%) genotypes. In conclusion, there were variations in ß-lactam resistance among Klebsiella spp. from health facilities situated in the northern, middle, and southern belts of Ghana. The study provides preliminary evidence that emphasizes the need to direct more attention to antimicrobial resistance control, especially in the northern belt of Ghana. Findings from this study may be critical for creating and fine-tuning effective antimicrobial resistance control strategies and for informing accurate antibiotic prescription by practitioners.