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.

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.

Cytotoxic and Antioxidant Effects of Antimalarial Herbal Mixtures.

Many developing countries depend on herbal mixtures as the first line and cost-effective therapy for malaria. These mixtures with such curative tendencies may also be a source of toxicity to host cells. On the other hand, these mixtures may have anticancer potential activity characterized by cytotoxicity to cancer cells. The aim of the study was to determine the cytotoxic and antioxidant effects of five different antimalarial herbal mixtures. Five antimalarial herbal mixtures commonly used in Ghana (coded as STF, SMH, SMM, SGM, and STT) were purchased and freeze-dried. The dried samples were tested on human acute T-cell leukemia (Jurkat) and breast adenocarcinoma (MCF-7) cell lines. Cytotoxicity was assessed using the tetrazolium-based colorimetric (MTT) assay while antioxidant activity was determined using DPPH free-radical scavenging assay. Among the mixtures, SMM and SGM exhibited the strongest cytotoxicity towards Jurkat cells (IC50 values 59.17 µg/ml and 49.57 µg/ml, respectively), whereas STT showed the weakest cytotoxicity (IC50 = 244.94 µg/ml). Cytotoxic effect of SMM was also strongest towards MCF-7 cells whilst the least cytotoxic sample was SGM (IC50 > 1000 µg/ml). SMM had the highest antioxidant percentage (EC50 = 1.05 mg/ml). The increasing order of antioxidant percentage among the five herbal mixtures is SMM > SMH > STT > STF > SGM. The herbal mixtures may be potential sources of toxic agents to host cells. Therefore, further toxicity studies must be performed to safeguard health of the public. Interestingly, cytotoxicities exhibited by SMM and SGM suggest the presence of anticancer constituents in them which warrant further studies.

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.

Diagnostic Yield of Fluorescence and Ziehl-Neelsen Staining Techniques in the Diagnosis of Pulmonary Tuberculosis: A Comparative Study in a District Health Facility.

BACKGROUND: Despite the recent advancement in diagnostic methods, the smear microscopy remains the gold standard for the diagnosis of pulmonary tuberculosis in high burden countries like Ghana. Notwithstanding, fluorescence staining technique provides a more efficient option for the detection of Mycobacterium tuberculosis positive smears. This study therefore aimed at assessing the diagnostic performance of fluorescence microscopy (FM) and Ziehl-Neelsen (ZN) staining techniques in the diagnosis of pulmonary tuberculosis. METHODS: A comparative study was carried out on 100 patients who reported at the Out Patients Department (OPD) or the Directly Observed Therapy (DOT) center of the Kade Government Hospital and were suspected of having pulmonary tuberculosis. Two (2) sputum samples each were collected. This included one spot and one morning sample. The smears were prepared and stained with FM and ZN staining techniques. Xpert MTB/RIF assay was also performed. RESULTS: Of the 200 samples analyzed, 71 (35.5%), 46 (23.0%), and 84 (42.0%) were positive for pulmonary tuberculosis when FM, ZN, and XPERT MTB/RIF assays were used, respectively. The mean reading time of FM was three times faster than the ZN technique with very good acceptance (1.5min: 4.6min). The sensitivity and specificity of fluorescent staining to that of XPERT MTB/RIF assay were 84.5% and 100%, respectively, while those of ZN staining were 54.8% and 100%, respectively. CONCLUSION: For a routine laboratory test in a resource-limited setting, our study has demonstrated that fluorescence staining technique is a more sensitive test for the diagnosis of pulmonary tuberculosis as compared to the conventional ZN technique.

Bispecific Antibodies (bsAbs): Promising Immunotherapeutic Agents for Cancer Therapy.

Antibodies have become the preferred therapeutic treatment option for cancers. Antibody therapy is associated with low toxic profile and specific in its activity, unlike chemotherapy and radiotherapy. Types of tumor are known to express multiple receptors that cross-talk to activate perpetual growth, proliferation and metastasis, and inhibit apoptosis in such tumors. Bispecific antibodies (BsAbs) are therefore the preferred agent for the treatment of such cancers due to its unique characteristics. This review discusses up to date therapeutic potentials of BsAbs.

5'-UTR of malS increases the invasive capacity of Salmonella enterica serovar Typhi by influencing the expression of bax.

AIM: An RNA-seq analysis recently identified a 236-nucleotide transcript upstream from malS in Salmonella enterica serovar Typhi. Here, we investigated its molecular characteristics and function. MATERIALS & METHODS: RACE and northern blotting were used to determine the molecular characteristics of the sequence, and mutagenesis, microarray, immunoblotting and an invasion assay were used to investigate the functions of the transcript. RESULTS: The transcript was identified as the malS 5'-untranslated region (UTR), which could influence the expression of the flagellar and SPI-1 genes and the invasion of HeLa cells by S. Typhi. Deletion of bax increased the expression of the invasion genes and the invasive capacity of S. Typhi, whereas the expression of the malS 5'-UTR reduced the expression of bax. CONCLUSION: The malS 5'-UTR reduces the expression of bax and increases the invasive capacity of S. Typhi.

Identification and characterization of a cis antisense RNA of the parC gene encoding DNA topoisomerase IV of Salmonella enterica serovar Typhi.

Bacterial cis-encoded antisense RNAs are transcribed from the opposite strand of protein coding genes, and their regulatory roles adapt cells to changing environmental conditions. By deep sequencing of the transcriptome of Salmonella enterica serovar Typhi, an antisense RNA that is encoded in cis to the parC gene was found. parC encodes the subunit A component of topoisomerase IV, a class of enzymes that relax both positively and negatively supercoiled DNA and are also required for segregation of daughter chromosomes in bacteria. Transcription of the 871 nucleotide antisense RNA was confirmed by northern blot and RACE analysis to be expressed mostly in the stationary phase of bacterial growth and also upregulated in iron limitation and osmotic stress conditions. Overexpression of the antisense RNA resulted in a significant increase in parC mRNA levels. Further analysis revealed that expression of the antisense RNA stabilizes the target mRNA, probably by protecting it from endoribonucleases. Our findings confirm and add to the ever increasing knowledge of the important role that regulatory antisense RNAs play in bacteria.

Identification and characterization of a cis-encoded antisense RNA associated with the replication process of Salmonella enterica serovar Typhi.

Antisense RNAs that originate from the complementary strand of protein coding genes are involved in the regulation of gene expression in all domains of life. In bacteria, some of these antisense RNAs are transcriptional noise while others play a vital role to adapt the cell to changing environmental conditions. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi, a partial RNA sequence encoded in-cis to the dnaA gene was revealed. Northern blot and RACE analysis confirmed the transcription of this antisense RNA which was expressed mostly in the stationary phase of the bacterial growth and also under iron limitation and osmotic stress. Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of dnaA, which will ultimately enhance their translation. Our findings have revealed that antisense RNA of dnaA is indeed transcribed not merely as a by-product of the cell's transcription machinery but plays a vital role as far as stability of dnaA mRNA is concerned.

Fis is essential for the stability of linear plasmid pBSSB1 and affects the motility of Salmonella enterica serovar Typhi.

pBSSB1 is a 27 kb non-bacteriophage-related linear plasmid first found in Salmonella enterica serovar Typhi (S. Typhi), but the mechanism underlying the replication of pBSSB1 is currently unknown. Previous reports showed that the factor for inversion stimulation (Fis) encoded by fis can affect the replication, transcription and other processes through binding DNA. Here, a fis deletion mutant of S. Typhi (Δfis) was prepared through the homologous recombination mediated by suicide plasmid and the loss of pBSSB1 in Δfis was observed surprisingly by pulsed field gel electrophoresis (PFGE). Subsequently, the loss of pBSSB1 was verified by PCR and Southern blot. In addition, the motility of Δfis was deficient and the flagellin of Δfis could not be detected by 2-dimensional polyacrylamide gel electrophoresis. All these results show that Fis is essential for the stability of pBSSB1 and affects the motility of S. Typhi.