Laboratory Isolates Research Articles

Borrelia PeptideAtlas: A proteome resource of common Borrelia burgdorferi isolates for Lyme research

Lyme disease is caused by an infection with the spirochete Borrelia burgdorferi, and is the most common vector-borne disease in North America. B. burgdorferi isolates harbor extensive genomic and proteomic variability and further comparison of isolates is key to understanding the infectivity of the spirochetes and biological impacts of identified sequence variants. Here, we applied both transcriptome analysis and mass spectrometry-based proteomics to assemble peptide datasets of B. burgdorferi laboratory isolates B31, MM1, and the infective isolate B31-5A4, to provide a publicly available Borrelia PeptideAtlas. Included are total proteome, secretome, and membrane proteome identifications of the individual isolates. Proteomic data collected from 35 different experiment datasets, totaling 386 mass spectrometry runs, have identified 81,967 distinct peptides, which map to 1,113 proteins. The Borrelia PeptideAtlas covers 86% of the total B31 proteome of 1,291 protein sequences. The Borrelia PeptideAtlas is an extensible comprehensive peptide repository with proteomic information from B. burgdorferi isolates useful for Lyme disease research.

Knockdown resistance in Stomoxys calcitrans stable fly populations on German dairy farms: kdr alleles explain susceptibility of individual flies to deltamethrin

AbstractThe stable fly, Stomoxys calcitrans, is a globally important pest causing stress, economic losses and transmission of pathogens in livestock. Control on commercial farms relies predominantly on the use of insecticides, with pyrethroids being the most frequently used class of insecticides in industrialised countries. Here, laboratory isolates were obtained from four dairy farms in Brandenburg (Germany) and tested for phenotypic resistance to deltamethrin in comparison to a susceptible reference isolate using topical application. Individual flies were subsequently genotyped using allele-specific real-time PCRs. Phenotypic resistance was observed in all four field isolates with resistance ratios between 46 and 119 compared to the susceptible laboratory strain. At position 1014 of the voltage-sensitive sodium channel, allele-specific PCRs detected the wild-type, kdr-his and kdr genotypes encoding leucine, histidine and phenylalanine, respectively. In the susceptible laboratory isolate, only the wild-type was identified. On the farms with very high LD50 values, the kdr variant was most prevalent and logistic regression analysis revealed that the kdr variant increased the odds to survive exposure to deltamethrin more than the kdr-his genotype. Flies carrying two resistance alleles were less susceptible than flies that also carried one wild-type allele. In three out of four field isolates, the allele frequencies were significantly different from the expectations of the Hardy–Weinberg equilibrium suggesting ongoing selection. The data show that the phenotype can be largely explained by the kdr genotype and represent high frequencies of the L1014F kdr and L1014H kdr-his variants conferring high levels of resistance in northern Germany.

Fidaxomicin resistance in Clostridioides difficile: a systematic review and predictive modeling with RNA polymerase binding sites.

Fidaxomicin (FDX), an RNA polymerase (RNAP) inhibitor antibiotic, is a guideline-recommended therapy for Clostridioides difficile infection. Mutations associated with reduced FDX minimum inhibitory concentrations (MICs) have been identified. However, the molecular characterization of these mutations on FDX binding and the development of FDX resistance have not been studied. The purpose of this systematic review was to identify FDX resistance in C. difficile isolates and determine whether single nucleotide polymorphisms associated with increased FDX MIC aligned with the RNAP binding pocket interacting residues. A systematic literature search was done in PubMed (1991-2023) with identified articles and their bibliographies searched for papers that included C. difficile genetic mutations and increased FDX MIC. Visualization of FDX-RNAP interactions was performed on Schrödinger Maestro using the publicly available C. difficile RNAP with fidaxomicin sequence (code 7L7B) on the Protein Data Bank. Seven articles were identified after applying inclusion and exclusion criteria. The most common mutation in clinical and laboratory isolates was at position V1143 of the β subunit, which accounted for approximately 50% of the identified mutations. Most other mutations occurred within the β' subunit of RNAP. Approximately one-third of the identified mutation aligned directly with FDX interacting residues with C. difficile RNAP (7/20) with most of the remainder occurring within 5 Å of the binding residues. C. difficile strains with elevated FDX MIC align closely with the known RNAP binding residues. These data demonstrate the potential to identify genomic methods to identify emerging FDX resistance.

MassARRAY: a high-throughput solution for rapid detection of foodborne pathogens in real-world settings.

Bacterial foodborne pathogens pose a substantial global public health concern, prompting government agencies and public health organizations to establish food safety guidelines and regulations aimed at mitigating the risk of foodborne illness. The advent of DNA-based amplification coupled with mass spectrometry, known as MassARRAY analysis, has proven to be a highly precise, sensitive, high-throughput, and cost-effective method for bacterial detection. This study aimed to develop, validate, and evaluate a MassARRAY-based assay for the detection and identification of significant enteropathogenic bacteria. The MassARRAY-based assay was developed for the detection of 10 crucial bacterial foodborne pathogens, including Campylobacter coli, Campylobacter jejuni, Clostridium perfringens, Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Salmonella spp., Shigella spp., and Staphylococcus aureus. The assay was optimized using the reference gDNA (n = 19), followed by validation using gDNA (n = 85) of reference and laboratory isolates. Additionally, the evaluation of the assay's reaction using a mixture of gDNA from all nine targeted species was performed. The limit of detection of the developed MassARRAY-based assay was determined using bacterial cells. Moreover, the validation method for field samples was evaluated by comparing it with standard microbiological testing methods routinely analyzed. The developed MassARRAY-based assay demonstrated 100% concordance with known bacterial pure cultures. The assay's reaction using a mixture of gDNA from all nine targeted species revealed the MassARRAY's capability to detect all targeted species in a single assay with the lowest concentration of 1 ng/μL of gDNA. The limits of detection of the assay range from 357 ± 101 to 282,000 ± 79,196 cells. Moreover, the validation of the assay in field samples revealed a 100% correlation between the data obtained from the standard microbiological method and the MassARRAY-based assay. These findings suggested that the developed MassARRAY-based assay exhibited the excellence in high-throughput detection of foodborne bacterial pathogens with high accuracy, reliability, and potential applicability within real-world field samples.

Genomic and phylogenetic features of the Picobirnaviridae suggest microbial rather than animal hosts.

The RNA virus family Picobirnaviridae has traditionally been associated with the gastrointestinal systems of terrestrial mammals and birds, with the majority of viruses detected in animal stool samples. Metatranscriptomic studies of vertebrates, invertebrates, microbial communities, and environmental samples have resulted in an enormous expansion of the genomic and phylogenetic diversity of this family. Yet picobirnaviruses remain poorly classified, with only one genus and three species formally ratified by the International Committee of Virus Taxonomy. Additionally, an inability to culture picobirnaviruses in a laboratory setting or isolate them in animal tissue samples, combined with the presence of bacterial genetic motifs in their genomes, suggests that these viruses may represent RNA bacteriophage rather than being associated with animal infection. Utilising a data set of 2,286 picobirnaviruses sourced from mammals, birds, reptiles, fish, invertebrates, microbial communities, and environmental samples, we identified seven consistent phylogenetic clusters likely representing Picobirnavirus genera that we tentatively name 'Alpha-', 'Beta-', 'Gamma-', 'Delta-', 'Epsilon-', 'Zeta-', and 'Etapicobirnavirus'. A statistical analysis of topological congruence between virus-host phylogenies revealed more frequent cross-species transmission than any other RNA virus family. In addition, bacterial ribosomal binding site motifs were more enriched in Picobirnavirus genomes than in the two groups of established RNA bacteriophage-the Leviviricetes and Cystoviridae. Overall, our findings support the hypothesis that the Picobirnaviridae have bacterial hosts and provide a lower-level taxonomic classification for this highly diverse and ubiquitous family of RNA viruses.

Evolution of H6N6 viruses in China between 2014 and 2019 involves multiple reassortment events

ABSTRACT H6N6 avian influenza viruses (AIVs) have been widely detected in wild birds, poultry, and even mammals. Recently, H6N6 viruses were reported to be involved in the generation of H5 and H7 subtype viruses. To investigate the emergence, evolutionary pattern, and potential for an epidemic of H6N6 viruses, the complete genomes of 198 H6N6 viruses were analyzed, including 168 H6N6 viruses deposited in the NCBI and GISAID databases from inception to January 2019 and 30 isolates collected from China between November 2014 and January 2019. Using phylogenetic analysis, the 198 strains of H6N6 viruses were identified as 98 genotypes. Molecular clock analysis indicated that the evolution of H6N6 viruses in China was constant and not interrupted by selective pressure. Notably, the laboratory isolates reassorted with six subtype viruses: H6N2, H5N6, H7N9, H5N2, H4N2, and H6N8, resulting in nine novel H6N6 reassortment events. These results suggested that H6N6 viruses can act as an intermediary in the evolution of H5N6, H6N6, and H7N9 viruses. Animal experiments demonstrated that the 10 representative H6N6 viruses showed low pathogenicity in chickens and were capable of infecting mice without prior adaptation. Our findings suggest that H6N6 viruses play an important role in the evolution of AIVs, and it is necessary to continuously monitor and evaluate the potential epidemic of the H6N6 subtype viruses.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

Protein isolates made in the laboratory versus the factory: A case study with mung bean and chickpea

AbstractProtein isolates deliver functionalities such as gelling, emulsifying, and foaming, and many recent publications are reporting the functionality of new protein isolates produced in the laboratory. Laboratory protein isolation processes are quite different to industrial processes, and therefore isolate functionalities are likely to be different. We examined some of the differences between laboratory isolates and commercial isolates of mung bean and chickpea protein (MPBI and CPPI). We tested solubility as a function of pH, dispersibility by static light scattering, and viscosity enhancement in a rapid visco analyzer (RVA). Thermal transitions were evaluated with differential scanning calorimetry (DSC), and powder morphology was examined by scanning electron microscopy (SEM). Laboratory isolates were more soluble (e.g. 54%–57% vs. 5%–45% at pH 8) and dispersible than commercial isolates, with the largest DSC endotherms, indicating the least denaturation. Among commercial isolates, we found large differences in solubility, thermal properties, and viscosity enhancement, and minor differences in dispersibility and powder morphology. The hydration protocol had a strong effect on solubility and DSC results, and a moderate effect on RVA results. Comparisons between isolates produced in different laboratories are currently hampered by the use of nonstandard empirical methods for certain functionalities, and we highlight standardized methods for measuring dispersibility and viscosity enhancement. Protein isolation processes affect isolate functionality via effects on protein denaturation and aggregation, and via the morphology and surface chemistry of isolate powders. This means that findings with laboratory isolates cannot necessarily be extrapolated to commercial counterparts but also indicates the power of process design to modulate isolate functionality.

Assessment of the Zoonotic Potential of Atypical Scrapie Prions in Humanized Mice Reveals Rare Phenotypic Convergence but Not Identity With Sporadic Creutzfeldt-Jakob Disease Prions.

Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from classical bovine spongiform encephalopathy (C-BSE) may copropagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, 1 isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. The results obtained suggest a low zoonotic potential for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.

A Comparative Genomic Analysis of Georgenia sps. for Mining of LysR Transcriptional Regulator Sequences

Georgenia is a genus belonging to the actinomycetes group. The genera comprise only 33 poorly characterized species with reference genomes of 10 distinct species. However, none of the species is well characterized for their genome characteristics. Our laboratory isolate from tomato plant leaf was identified and sequenced for identification and found to be Georgenia sp. Later genomic analysis revealed many functional genes having characteristic functions to be analysed. The source of isolation raised the possibility of having functional genes to enhance senescence or having plant pathogenic activity by Georgenia sp. SUBG003. To explore in silico presence of these genes or gene pools genomic islands were identified and analysed for our isolate and other 10 reference genomes of the Georgenia genus. Genomic islands were further explored for transcription regulators and finally, LysR transcriptional regulator sequences were extracted and a phylogeny among sequences was built from multiple sequence alignment.

Prevalence of Salmonella spp. and Escherichia coli in the feces of free-roaming wildlife throughout South Korea.

Wildlife can carry pathogenic organisms, including viruses, bacteria, parasites, and fungi, which can spread to humans and cause mild to serious illnesses and even death. Spreading through animal feces, these pathogens significantly contributes to the global burden of human diseases. Therefore, the present study investigated the prevalence of zoonotic bacterial pathogens, such as Salmonella spp., Escherichia coli, and Shiga toxin-producing E. coli (STEC), in animal feces. Between September 2015 and August 2017, 699 wildlife fecal samples were collected from various agricultural production regions and mountainous areas in South Korea. Fecal samples were collected from wild mammals (85.26%, 596/699) and birds (14.73%, 103/699). Salmonella spp. and E. coli were present in 3% (21/699) and 45.63% (319/699) of the samples, respectively. Moreover, virulence genes stx1 and both stx1 and stx2 were detected in 13.30% (93/699) and 0.72% (5/699) of the samples, respectively. The 21 Salmonella spp. were detected in badgers (n = 5), leopard cats (n = 7), wild boars (n = 2), and magpies (n = 7); STEC was detected in roe deer, water deer, mice, and wild boars. Through phylogenetic and gene-network analyses, the Salmonella spp. isolates (n = 21 laboratory isolates, at least one isolate from each Salmonella-positive animal fecal sample, and n = 6 widely prevalent reference Salmonella serovars) were grouped into two major lineages: S. enterica subsp. enterica and S. enterica subsp. diarizonae. Similarly, 93 E. coli isolates belonged to stx1, including three major lineages (groups 1-3), and stx1 and stx2 detected groups. To the best of our knowledge, this is the first report of a wild leopard cat serving as a reservoir for Salmonella spp. in South Korea. The research findings can help manage the potential risk of wildlife contamination and improve precautionary measures to protect public health.

Extended-spectrum beta-lactamase-producing Enterobacterales in patients with suspected sepsis in an acute care setting in Skåne, Sweden: a cohort study

Introduction Epidemiological data on extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales are most often based on microbiological laboratory isolates and do not consider important clinical data such as infection or colonisation, treatment, and outcome. This study aimed to assess prevalence of ESBL-producing Enterobacterales as the cause of infection in patients with suspected sepsis in the emergency department based on clinical data. It also examined the number of patients with suspected sepsis who had ESBL-producing pathogens, comparing estimates that were based on laboratory data versus a combination of laboratory and clinical data. Methods Patients with suspected sepsis in the emergency department at Skåne University Hospital, Lund, Sweden were included consecutively. Data were collected retrospectively from medical records. Results Of the 764 included patients, 223 patients had growth of Enterobacterales in any specimen (i.e. colonisation or infection according to laboratory data), while 191 patients had Enterobacterales detected in the blood or in the suspected focus of infection (i.e. an infection according to clinical and laboratory data). Eighteen patients had ESBL-producing Enterobacterales in any clinical specimen, 11 of whom had an infection with ESBL-producing Enterobacterales, resulting in a prevalence of infections with ESBL-producing Enterobacterales in infected patients with suspected sepsis of 1.8%. The number of patients with ESBL-producing Enterobacterales was not significantly different when infection was defined using laboratory data alone versus a combination of laboratory and clinical data [18/223 (8.1%) vs 11/191 (5.8%), p = 0.36]. Conclusions The prevalence of ESBL-producing Enterobacterales infections among patients with suspected sepsis is low in an acute care setting in Sweden.

A cell-free strategy for host-specific profiling of intracellular antibiotic sensitivity and resistance

Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease-causing microbes. To provide a host-specific tool to study antibiotic susceptibility and resistance, here we develop Klebsiella pneumoniae cell-free gene expression (CFE) systems from laboratory and clinical isolates. Using proteomics, we identify relative differences and unique proteins for these new CFE systems in comparison to an Escherichia coli MG1655 CFE model. Then we profile antimicrobial susceptibility in parallel with whole cells to quantify CFE antibiotic potency. Finally, we apply this native CFE tool to study AMR variants at a proof-of-concept level. Definably we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin—a genotype observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. Overall, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial extract processing requires Biosafety Level 2, the CFE system is non-living, suitable for long-term storage and study in a Biosafety Level 1 lab. We anticipate the K. pneumoniae CFE bioassay is advantageous for host-specific antimicrobial testing, the characterisation of intracellular AMR variants and potentially structure-activity relationship studies.

Biotic potential and pathogenicity of a Haemonchus contortus susceptible laboratory isolate compared to a resistant field isolate in Brazil

The high prevalence of anthelmintic resistance of Haemonchus contortus is threatening sheep farming. In this context, resistant populations’ replacement using susceptible isolates appears to be a strategy to overcome this situation. The aim of the present study was to evaluate the ability of a susceptible H. contortus laboratory isolate in comparison with a multiresistant field isolate to infect lambs and to study its behaviour in field conditions. Sixteen worm-free lambs were experimentally infected with 4000 infective larvae (L3) of a H. contortus anthelmintic-resistant field isolate (Group R, n = 8) or a laboratory isolate susceptible to anthelmintics (Group S, n = 8). Groups were placed in separate clean pastures, where they were kept for 266 days. Faecal egg counts (FEC) showed maximum values between 21- and 42-days post-infection (PI) and decreased over time, with no statistical differences between groups (P > 0.05). The pathogenicity of the isolates was similar, as lambs from both groups showed a decrease in packed cell volume and total plasma protein values in the beginning of the study, coinciding with the highest FECs. Production of L3 were similar for both groups, when evaluating faecal cultures kept in vitro or L3 recovered from pasture samples and from faecal pats collected from the paddocks, which suggests that both isolates have the same potential of development in vitro and in the environment. The degree of resistance/susceptibility to thiabendazole (TIA), levamisole (LEV), monepantel (MON) and ivermectin (IVE) was evaluated using the Larval Development Assay (RESISTA-Test©) 160 days PI. The resistant isolate showed a high resistance factor (RF) to TIA (141), LEV (822), MON (34) and IVE (196), while the RF of the susceptible isolate remained below three, which indicates high susceptibility to all drugs tested. Our study demonstrates that the susceptible isolate was able to infect lambs and did not show higher pathogenicity nor any differences on its biotic potential when compared to the resistant field isolate. These results indicate that the susceptible isolate has the potential to be used to replace a resistant population of H. contortus, which should be validated in long-term studies under commercial sheep farming conditions.

Discovery and genome-guided mapping of REN12 from Vitis amurensis, conferring strong, rapid resistance to grapevine powdery mildew.

Powdery mildew resistance genes restrict infection attempts at different stages of pathogenesis. Here, a strong and rapid powdery mildew resistance phenotype was discovered from Vitis amurensis 'PI 588631' that rapidly stopped over 97% of Erysiphe necator conidia, before or immediately after emergence of a secondary hypha from appressoria. This resistance was effective across multiple years of vineyard evaluation on leaves, stems, rachises, and fruit and against a diverse array of E. necator laboratory isolates. Using core genome rhAmpSeq markers, resistance mapped to a single dominant locus (here named REN12) on chromosome 13 near 22.8-27.0Mb, irrespective of tissue type, explaining up to 86.9% of the phenotypic variation observed on leaves. Shotgun sequencing of recombinant vines using skim-seq technology enabled the locus to be further resolved to a 780kb region, from 25.15 to 25.93Mb. RNASeq analysis indicated the allele-specific expression of four resistance genes (NLRs) from the resistant parent. REN12 is one of the strongest powdery mildew resistance loci in grapevine yet documented, and the rhAmpSeq sequences presented here can be directly used for marker-assisted selection or converted to other genotyping platforms. While no virulent isolates were identified among the genetically diverse isolates and wild populations of E. necator tested here, NLR loci like REN12 are often race-specific. Thus, stacking of multiple resistance genes and minimal use of fungicides should enhance the durability of resistance and could enable a 90% reduction in fungicides in low-rainfall climates where few other pathogens attack the foliage or fruit.

Growth Response of Oil Palm Plant Seeds after Application of Vesicular Arbuscular Mycorrhiza (VAM) Fungus

Objectives: This study aims to determine the growth response of oil palm seedlings to VAM and to determine the appropriate VAM dose for oil palm seedling growth. Methods: This study took place in the screen house and plant protection laboratory and VAM isolate starters were gathered from the work area. The test used a factorial completely randomized design (CRD) with 2. The first treatment was VAM dose with 4 factors and the second was NPK fertilizer with two factors. The VAM was applied for one month after germination and plant maintenance for 16 weeks with seven parameters. Findings: The results show that the VAM dose treatment and the application of NPK fertilizer did not have a significant effect on the growth of oil palm seedlings in the pre-nursery (16 weeks) so it took a long time to see the role of VAM in oil palm seedlings. The application of VAM 150 g/polybag on oil palm seedlings in pre-nursery tended to give the best response to the growth of seedling height, a number of leaves, fresh weight of the crown, dry weight of the crown, fresh weight of roots, dry weight of roots and root volume. The VAM inoculum supplied made symbiosis with oil palm seedling roots with a high infection rate. The number of VAM spores in the media and the percentage of colonization on the roots were influenced by the dose of VAM inoculum given. Novelty: Vesicular Arbuscular Mycorrhiza (VAM) Fungus in oil palm seeds are important to increase the growth of the number of leaves, fresh weight of the crown, and dry weight of the crown. Efforts to increase the quality and quantity of oil palm production are appropriately necessary so that the desired target can be achieved. Keywords: Growth Response; Oil Palm Seeds; VAM Fungus; Number of Leaves; Fresh Weight of the crown

Bacterial sensitivity to chlorhexidine and povidone-iodine antiseptics over time: a systematic review and meta-analysis of human-derived data

Surgical site infection (SSI) is the most common complication of surgery, increasing healthcare costs and hospital stay. Chlorhexidine (CHX) and povidone-iodine (PVI) are used for skin antisepsis, minimising SSIs. There is concern that resistance to topical biocides may be emergeing, although the potential clinical implications remain unclear. The objective of this systematic review was to determine whether the minimum bactericidal concentration (MBC) of topical preparations of CHX or PVI have changed over time, in microbes relevant to SSI. We included studies reporting the MBC of laboratory and clinical isolates of common microbes to CHX and PVI. We excluded studies using non-human samples and antimicrobial solvents or mixtures with other active substances. MBC was pooled in random effects meta-analyses and the change in MBC over time was explored using meta-regression. Seventy-nine studies were included, analysing 6218 microbes over 45 years. Most studies investigated CHX (93%), with insufficient data for meta-analysis of PVI. There was no change in the MBC of CHX to Staphylococci or Streptococci over time. Overall, we find no evidence of reduced susceptibility of common SSI-causing microbes to CHX over time. This provides reassurance and confidence in the worldwide guidance that CHX should remain the first-choice agent for surgical skin antisepsis.

Antibacterial Activity of Ampelocissus latifolia Tuberous Root Extract against Diverse Microbes

Microbial drug resistance is growing every day and novel drugs are need of the hour to counter the future threat of large pandemic. The present work for the first time explores the antibacterial activity of methanol, hydro-alcoholic and n-hexane extracts of Ampelocissus latifolia (A. latifolia) tuberous root against few laboratory and clinical isolates. A total of 10 microbes were screened; out of which 1 is a laboratory strain (acid-fast) and the rest 9 are clinical isolates (5 Gram-positive and 4 are Gram-negative), 2 clinical isolates were drug-resistant (1 Gram-positive and 1 Gram-negative). In this screening, methanol extract showed potential antibacterial activity against Gram-positive, Gram-negative and acid-fast bacteria with zones of inhibition measuring 18 mm, 10 mm and 8 mm, respectively. Hydro-alcoholic and n-hexane extract showed a smaller zone of inhibition compared to methanol extract and the positive control. The minimum inhibitory concentration (MIC) of methanol and the hydro-alcoholic extract of powdered tuber root was determined by the micro-broth dilution method. The minimum inhibitory concentration of methanol and hydro-alcoholic extract for Gram-positive and Gram-negative microbes varies from 0.18 mg/mL to 1.5 mg/mL. The methanol and hydro-alcoholic extracts were analyzed with FTIR, which revealed the presence of alcohol, alkanes, alkenes, alkyls, ketones and nitro groups in the methanol and hydro-alcoholic extracts of A. latifolia tuberous root. The high-performance thin-layer chromatography (HPTLC) analysis of the methanol extract indicates the presence of at least 5 different compounds with different Rf values.

Differential antimicrobial extract activity of Graviola (Annona muricata) on gram positive and gram- negative antibiotic-resistant bacteria

New agents are needed with the increasing prevalence of multidrug-resistant (MDR) bacteria. Identification of plants with activity against MDR bacteria is of increasing importance. The antimicrobial activity of commercially available Graviola preparations against 38 clinical and laboratory isolates, representing MDR and drug-sensitive Gram positive and Gram negative bacteria, was measured by broth microdilution. Commercially Graviola fruit, as well as leaves/stems preparations, exhibited both inhibitory and cidal activity. However, the fruit preparation showed significantly less activity than the leaves/stems preparation. The Graviola leaves/stems preparation activity titer ranged from 1:4 to 1:16 against MDR Gram positive and Gram negative bacteria; including methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL) Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae carbapenemase (KPC). However, the preparation had no effect on Lactobacillus. In addition, further extractions of the leaves/stems were tested against MRSA and MSSA in an in-vitro biofilm wound infection model. The polar extracts of the stems/leaves exhibited the highest bactericidal activity in preventing and inhibiting biofilm formation. Thus, Graviola leaves/stems extracts showed differential activity for phytochemical utility against multidrug-resistant bacteria.

Whole Genome Sequencing of Antibiotic Resistant Genes in Isolates from Surfaces in a Science Laboratory.

Isolates obtained from laboratory surfaces were identified and characterized. Ten consecutive isolates were obtained from 30 sample surfaces of a University Science Laboratory in Edo State Nigeria in May, 2021. Swabs of surfaces from the laboratory were obtained aseptically. The sample swabs were streaked on MacConkey, eosin methylene blue, mannitol salt, and nutrient agar plates, respectively, and incubated appropriately. Distinct colonies were randomly obtained from culture plates and characterized phenotypically. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to analyze four isolates (40%) obtained by selection criteria. Susceptibility testing using antibiotics was performed for the identified isolates by Kirby-Bauer method for 15 antibiotics. Isolate characterization and identification of resistance determinants were determined using whole genome sequencing (WGS). Microorganisms identified included Leclercia adecarboxylata, Enterobacter hormaechei, Atlantibacter hermanii, and Stenotrophomonas maltophilia. Three identified isolates were antibiotics-resistant and were investigated by WGS. Resistance genes were found in all (100%) of the resistant laboratory isolates. The resistance determinants included β-lactamase genes, aminoglycoside modifying enzymes, qnr genes, sulfonamide, tetracycline, and trimethoprim resistance genes, respectively. Two isolates carried ESBL genes and blaCTX-M-15 was detected. Our study displays the dissemination of antibiotic resistance among isolates obtained from surface of a University Science Laboratory. To the best of our knowledge, we have reported the first genomic characterization of resistance to antibiotics in isolates obtained from surfaces of a University Science Laboratory in Nigeria.