Mouse Lethality Research Articles

Diagnosis of botulism in mammals aided by toxin ELISA and C and D gene RT-PCRs with an emphasis on farm animals.

Animal welfare and ethical considerations limit the use of the mouse lethality bioassay for the detection of botulinum toxins. This study assessed an ELISA and RT-PCRs to detect toxin types C and D in samples from mammals with flaccid paralysis or weakness. DNA/RNA tightly bound to the toxin molecules were used as surrogates for the presence of toxin. In total 391 diverse samples from 101 clinical cases from cattle and 9 other species were examined. Botulism was confirmed by ELISA in samples from 16 cases. Toxin type C or D was identified by RT-PCR in 10 cases, five of which were among the cases confirmed by ELISA. Among the 80 cases with negative samples, sampling was not comprehensive enough in 66 cases (botulism therefore not excluded) but was adequate in 14 cases (no indication of botulism). The last two categories included 16 cases with other laboratory findings explaining the clinical signs. The ELISA that detects botulinum C and D toxins was superior in identifying cases with positive samples compared to the RT-PCRs but the latter were able to identify the toxin type present when positive. Furthermore, the RT-PCRs are available to any laboratory with molecular testing capability. Both laboratory assays can screen a comprehensive range and a large number of samples which is crucial for a successful diagnosis of botulism.

Assessment of the Artemia salina toxicity assay as a substitute of the mouse lethality assay in the determination of venom-induced toxicity and preclinical efficacy of antivenom

Assessment of the Artemia salina toxicity assay as a substitute of the mouse lethality assay in the determination of venom-induced toxicity and preclinical efficacy of antivenom

Novel insights into the application of recombinant mutated phospholipases D as antigens for developing new strategies against Loxoscelism

Loxoscelism is the pathological condition triggered by a brown spider bite. The venom of these spiders is rich in phospholipases D (PLDs), which can induce virtually all local and systemic manifestations. Recombinant mutated PLDs from clinically relevant Loxosceles species in South America have been investigated as potential antigens to develop novel therapeutic strategies for loxoscelism. However, certain gaps need to be addressed before a clinical approach can be implemented. In this study, we examined the potential of these recombinant mutated PLDs as antigens by testing some variations in the immunization scheme. Furthermore, we evaluated the efficacy of the produced antibodies in neutralizing the nephrotoxicity and sphingomyelinase activity of brown spider venoms. Our findings indicate that the number of immunizations has a greater impact on the effectiveness of neutralization compared to the amount of antigen. Specifically, two or three doses were equally effective in reducing dermonecrosis and edema. Additionally, three immunizations proved to be more effective in neutralizing mice lethality than one or two. Moreover, immunizations mitigated the signs of kidney injury, a crucial aspect given that acute renal failure is a serious systemic complication. In vitro inhibition of the sphingomyelinase activity of Loxosceles venoms, a key factor in vivo toxicity, was nearly complete after incubation with antibodies raised against these antigens. These findings underscore the importance of implementing an effective immunization scheme with multiple immunizations, without the need for high antigen doses, and enhances the spectrum of neutralization exhibited by antibodies generated with these antigens. In summary, these results highlight the strong potential of these antigens for the development of new therapeutic strategies against cutaneous and systemic manifestations of loxoscelism.

Assessment of the Artemia salina toxicity assay as a substitute of the mouse lethality assay in the determination of venom-induced toxicity and preclinical efficacy of antivenom

Mice are routinely used in snake venom research but are costly and subject to pain and suffering. The crustacean Artemia salina could be an alternative to mice, but data to support its adoption in snake venom research is limited. The aim of the present study was to evaluate the suitability of A. salina as a surrogate of mice in assessing the toxicity of venoms and the preclinical efficacy of antivenoms. The toxicity of venoms from 22 snakes of medical importance in sub–Saharan Africa was evaluated in mice (intraperitoneally; i.p. and intravenously; i.v.) and in A. salina. Subsequently, the capacity of a commercial antivenom to neutralize the toxicity of these venoms in mice and A. salina was investigated. There was a positive correlation between the i.v. median lethal doses (LD50s) and the i.p. LD50s in mice (r = 0.804; p < 0.0001), a moderate correlation between the i.v. LD50s in mice and the median lethal concentrations (LC50s) in A. salina (r = 0.606; p = 0.003), and a moderate correlation between the i.p. LD50s in mice and the LC50s in A. salina (r = 0.426; p = 0.048). Moreover, there was a strong correlation between the i.p. median effective doses (ED50s) and the i.v. ED50s in mice (r = 0.941, p < 0.0001), between the i.p. ED50s in mice and the ED50s in A. salina (r = 0.818, p < 0.0001), and between the i.v. ED50s in mice and the ED50s in A. salina (r = 0.972, p < 0.0001). These findings present A. salina as a promising candidate for reducing reliance on mice in snake venom research. Future investigations should build upon these findings, addressing potential limitations and expanding the scope of A. salina in venom research and antivenom development.

О расчете неопределенности показателя токсичности продуктов горения полимерных материалов

A testing lab must evaluate and determine the contribution of the uncertainty of measurements for each testing methodology in order to implement the reliability and accuracy of tests. In addition, the uncertainty index must be specified in the test (measurement) protocols. Uncertainty can be calculated by using various methods; however, when the desired value of the output parameter is determined from a graph, the method of least squares should be used. In order to determine the toxicity index of combustion products, a series of tests using white laboratory mice have been conducted. Based on the test results, a graph of the dependence of lab mice lethality on the concentration of toxic combustion products has been built; the dose causing the lethality of 50 % of lab mice has been determined. Using the method of least squares, confidence limits of the estimate of measured value have been calculated and graphically shown. As a result of calculations, the uncertainty of toxicity of combustion products was determined as around 25 %. The wide range of data has been caused by the error of measuring equipment, heterogeneity of the test sample and its composition, as well as the characteristics of lab mice and their biological features. The results of uncertainty calculation provided in the article can be used by laboratories conducting fire safety tests of materials.

Genomic surveillance indicates clonal replacement of hypervirulent Klebsiella pneumoniae ST881 and ST29 lineage strains in vivo.

The emergence of hypervirulent Klebsiella pneumoniae (hvKp) poses a significant public health threat, particularly regarding its carriage in the healthy population. However, the genomic epidemiological characteristics and population dynamics of hvKp within a single patient across distinct infection episodes remain largely unknown. This study aimed to investigate the clonal replacement of hvKp K2-ST881 and K54-ST29 lineage strains in a single patient experiencing multiple-site infections during two independent episodes. Two strains, designated EDhvKp-1 and EDhvKp-2, were obtained from blood and cerebrospinal fluid during the first admission, and the strain isolated from blood on the second admission was named EDhvKp-3. Whole-genome sequencing, utilizing both short-read Illumina and long-read Oxford Nanopore platforms, was conducted. In silico multilocus sequence typing (MLST), identification of antimicrobial resistance and virulence genes, and the phylogenetic relationship between our strains and other K. pneumoniae ST881 and ST29 genomes retrieved from the public database were performed. Virulence potentials were assessed through a mouse lethality assay. Our study indicated that the strains were highly susceptible to multiple antimicrobial agents. Plasmid sequence analysis confirmed that both virulence plasmids, pEDhvKp-1 (166,008 bp) and pEDhvKp-3 (210,948 bp), belonged to IncFIB type. Multiple virulence genes, including rmpA, rmpA2, rmpC, rmpD, iroBCDN, iucABCD, and iutA, were identified. EDhvKp-1 and EDhvKp-2 showed the closest relationship to strain 502 (differing by 51 SNPs), while EDhvKp-3 exhibited 69 SNPs differences compared to strain TAKPN-1, which all recovered from Chinese patients in 2020. In the mouse infection experiment, both ST881 EDhvKp-1 and ST29 EDhvKp-3 displayed similar virulence traits, causing 90 and 100% of the mice to die within 72 h after intraperitoneal infection, respectively. Our study expands the spectrum of hvKp lineages and highlights genomic alterations associated with clonal switching between two distinct lineages of hvKP that successively replaced each other in vivo. The development of novel strategies for the surveillance, diagnosis, and treatment of high-risk hvKp is urgently needed.

RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae

ABSTRACT Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide, especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in K. pneumoniae. More than 3,000 TA loci in 2,000 K. pneumoniae plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent K. pneumoniae. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid- carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT, which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumoniae.

Drug Resistance and Molecular Characteristics of Carbapenem-Resistant OXA-48-Producing Klebsiella pneumoniae Strains in Hainan, China.

The emergence and global spread of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) are of great concern to health services worldwide. These β-lactamases hydrolyze almost all β-lactams, are plasmid-encoded, and are easily transferable among bacterial species. They are mostly of the KPC types in CR-hvKp. OXA-48-producing hvKP strains have been rarely reported in the literature. OXA-48-producing hvKP strains were collected from clinical specimens at the First Affiliated Hospital of Hainan Medical University from January 2022 to March 2023. Hypervirulent strains were tested for virulence in a mouse lethality study and underwent whole genome sequencing to identify genomic features. A total of 42 unique OXA-48-bearing K. pneumoniae strains were identified, including three CR-hvKP strains (KP2683-1, NCRE61, and KP2185), which were isolated from bacteremia, pulmonary abscess, and liver abscess separately. The three CR-hvKP strains belonged to two different clones of ST11 KL64 (KP2185 and NCRE61) and ST23 K1 (KP2683-1). The KP2683-1 strain had the highest virulence. Whole genome sequencing analysis indicated that NCRE61 and KP2185 acquired IncFIB-type plasmids with a set of virulence genes (iroBCDN, iucABCD, iutA, rmpA, and rmpA2), while KP2683-1 acquired an IncL-type blaOXA-48-harboring plasmid. Consecutive cultures showed that the blaOXA-48-harboring plasmids were highly stable in the three hvKP strains and could be transmitted to Escherichia coli J53 by conjugation. The drug susceptibility testing results show that Ceftazidime/avibactam is sensitive for OXA-48-producing hvKP. Our study highlighted the two evolutionary pathways of OXA-48-producing hvKP strains and confirmed their virulence through in vivo testing. Ceftazidime/avibactam may be a viable option for treating OXA-48-producing hvKP strains.

Recent understanding of botulinum toxin and its main detection methods

The Botulinum neurotoxin (BoNT) has the title of the most potent poison known to man. It causes a serious condition known as botulism. Guidelines and regulations for the industrial processing of food have made foodborne botulism rarer, but the use of the toxin in pharmaceutical industries and its potential as a bioweapon make detection methods of the toxin a necessary thing. Currently, the Mouse Lethality Bioassay (MLB) is still considered the golden standard of botulinum toxin detection even though it has several limitations. There had subsequently been the development of several alternatives to the MLB, including immunoassays, endopeptidase-based assays, and more. This paper gives an overview of the mechanisms behind botulinum toxins, current methods of detection, and the methods advantages and disadvantages.

Toxicological and morphological aspects of nano-TiO2 and nano-TiO2-Ag acute action on the liver of mice

Aim. To study the toxic effect of TiO2 and TiO2-Ag nanopowders on the morphology and elemental composition of the laboratory mice liver. Materials and methods. The study used a model of acute intoxication on laboratory animals. Mice were injected intraperitoneally with TiO2 and TiO2-Ag nanopowders at 4000 mg/kg, 7000 mg/kg, or 10000 mg/kg doses. During two weeks, the animals were observed, lethality was assessed, the accumulation of nanopowder in the organ and the morphology of liver tissues were investigated. The content of titanium and silver in liver samples was determined by optical emission spectroscopy with inductively coupled plasma. Liver tissue micropreparations were examined using an Olympus BX51 light microscope. Also, the micropreparations of the liver were examined by scanning electron microscopy (SEM) using the Tescan Mira 3 device, and the elemental composition was determined using an energy dispersive spectrometer Oxford instrument, X-max 80 mm2. Results. The dependence of the mice lethality on the nanopowders dose was revealed; mortality was higher when exposed to nano-TiO2-Ag compared to nano-TiO2. Average lethal doses were calculated using probit analysis. For nano-TiO2, the LD50 is 4783.30 mg/kg; for nano-TiO2-Ag – 724.44 mg/kg. The accumulation of titanium, titanium, and silver in the liver after exposure to nano-TiO2 and nano-TiO2-Ag was established. In general, there was a tendency to increase the content of titanium in the skin tissue with an increase in the administered dose of nanopowders. Morphological changes in the liver were studied by histological methods. The most characteristic morphological signs of the toxic effect of nano-TiO2 on tissue were dystrophic changes at the level of 67.7 % (cytoplasmic vacuolization in hepatocytes), and when exposed to nano-TiO2-Ag – initial necrotic changes at the level of 70 % (hepatocytes with nuclear pyknosis). It is worth noting that the toxic effect of nano-TiO2 and nano-TiO2-Ag is much less often manifested by focal necrosis and inflammatory reactions (focal infiltration), in some cases, there were adaptive changes that provoked an increase in the number of binuclear hepatocytes. In case of detection agglomerates of a foreign object (crystalline inclusions) were obtained, which were examined spectrally and showed a high content of titanium (Ti). SEM morphometry showed that the size of nanoparticles and their agglomerates ranged from 80 nm to 20 μm. Conclusions. The lethality of mice was higher when the composition of nano-TiO2-Ag was introduced compared to nano-TiO2. Based on the calculated average lethal doses, both nanopowders were assigned to the 3rd class (moderately dangerous) of the danger of chemical substances according to the classification of GOST 12.1.007-76. It was established that with an increase in the injected dose in the tissue of the products of laboratory mice, the accumulation of titanium (under the action of nano-TiO2) and titanium and silver (under the action of nano-TiO2-Ag) increases. Characteristic microscopic signs of the toxic effect of TiO2 and TiO2-Ag nanopowders after intraperitoneal injection in laboratory bags are dystrophic changes in hepatocytes, necrosis of parenchymal disease, while inflammatory reactions occur less often. SEM and the method of elemental mapping of titanium confirmed the presence of TiO2 nanoparticles and their agglomerates in skin tissue when TiO2 nanopowder was administered.

Analysis of an IncR plasmid carried by carbapenem-resistant Klebsiella pneumoniae: A survey of swine Klebsiella pneumoniae in Jilin Province

This study was conducted in Jilin Province to investigate the mechanism involved in the antibiotic resistance and pathogenicity of Klebsiella pneumoniae. Lung samples were collected from large-scale pig farms in Jilin Province. Antimicrobial susceptibility and mouse lethality assays were carried out. K. pneumoniae isolate JP20, with high virulence and antibiotic resistance, was chosen for whole-genome sequencing. The complete sequence of its genome was annotated, and the virulence and antibiotic resistance mechanism were analysed. A total of 32 K. pneumoniae strains were isolated and tested for antibiotic resistance and pathogenicity. Among them, the JP20 strain showed high levels of resistance to all tested antimicrobial agents and strong pathogenicity in mice (lethal dose of 1.35×1011 CFU/mL). Sequencing of the multidrug-resistant and highly virulent K. pneumoniae JP20 strain revealed that the antibiotic resistance genes were mainly carried by an IncR plasmid. We speculate that extended-spectrum β-lactamases and loss of outer membrane porin OmpK36 play an important role in carbapenem antibiotic resistance. This plasmid contains a mosaic structure consisting of a large number of mobile elements. Through genome-wide analysis, we found that an lncR plasmid carried by the JP20 strain may have evolved in pig farms, possibly leading to multidrug resistance in the JP20 strain. It is speculated that the antibiotic resistance of K. pneumoniae in pig farms is mainly mediated by mobile elements (insertion sequences, transposons, and plasmids). These data provide a basis for monitoring the antibiotic resistance of K. pneumoniae and lay a foundation for an improved understanding of the genomic characteristics and antibiotic resistance mechanism of K. pneumoniae.

Hypercapsule is the cornerstone of Klebsiella pneumoniae in inducing pyogenic liver abscess.

To investigate the mechanisms of Klebsiella pneumoniae-induced pyogenic liver abscess (PLA). Forty-three K. pneumoniae strains from PLAs and 436 from non-PLAs were collected. Their differences were compared for virulence genes and factors, sequence types, and serotypes. Virulence genes wzi, wzy-K1, and wzi+wzy-K1 were deleted in K. pneumoniae NTUH-K2044. Various analyses, such as transmission electron microscopy, neutrophil killing tests, and mouse lethality tests, were used to confirm the consequent changes. Differences were found between K. pneumoniae strains from PLA and non-PLA samples for virulence genes and factors, including metabolism genes (allS and peg-344), capsular polysaccharide (CPS)-synthesis channel gene (wzy-K1), CPS-regulating genes (p-rmpA, p-rmpA2, and c-rmpA), and siderophore genes (iucA and iroN). When wzy-K1 was positive, the difference between PLA and non-PLA samples was only observed with c-rmpA. Δwzi, Δwzy-K1, and ΔwziΔwzy-K1 strains reverted to hypovirulence. In the Kupffer cell stimulation assay, interleukin (IL)-6, IL-12, IL-10, and transforming growth factor-β secretions were found to be equivalent in NTUH-K2044, Δwzi, Δwzy-K1, and ΔwziΔwzy-K1 groups. Lower IL-1β and higher tumor necrosis factor-α secretions were observed for Δwzi, Δwzy-K1, and ΔwziΔwzy-K1. Hypercapsule production is the cornerstone of hypervirulence, regardless of exopolysaccharides. K1 K. pneumoniae-induced PLA may decrease core inflammatory cytokines rather than increase anti-inflammatory cytokines. Exopolysaccharides could also attenuate the inflammatory response to aid in the immune escape of K. pneumoniae.

Hypervirulent Klebsiella pneumoniae Causing Neonatal Bloodstream Infections: Emergence of NDM-1-Producing Hypervirulent ST11-K2 and ST15-K54 Strains Possessing pLVPK-Associated Markers.

Klebsiella pneumoniae is a major cause of neonatal sepsis. Hypervirulent Klebsiella pneumoniae (hvKP) that cause invasive infections and/or carbapenem-resistant hvKP (CR-hvKP) limit therapeutic options. Such strains causing neonatal sepsis have rarely been studied. Characterization of neonatal septicemic hvKP/CR-hvKP strains in terms of resistance and virulence was carried out. Antibiotic susceptibility, molecular characterization, evaluation of clonality, in vitro virulence, and transmissibility of carbapenemase genes were evaluated. Whole-genome sequencing (WGS) and mouse lethality assays were performed on strains harboring pLVPK-associated markers. About one-fourth (26%, 28/107) of the studied strains, leading to mortality in 39% (11/28) of the infected neonates, were categorized as hvKP. hvKP-K2 was the prevalent pathotype (64.2%, 18/28), but K54 and K57 were also identified. Most strains were clonally diverse belonging to 12 sequence types, of which ST14 was most common. Majority of hvKPs possessed virulence determinants, strong biofilm-forming, and high serum resistance ability. Nine hvKPs were carbapenem-resistant, harboring blaNDM-1/blaNDM-5 on conjugative plasmids of different replicon types. Two NDM-1-producing high-risk clones, ST11 and ST15, had pLVPK-associated markers (rmpA, rmpA2, iroBCDEN, iucABCDiutA, and peg-344), of which one co-transferred the markers along with blaNDM-1. The 2 strains revealed high inter-genomic resemblance with the other hvKP reference genomes, and were lethal in mouse model. To the best of our knowledge, this study is the first to report on the NDM-1-producing hvKP ST11-K2 and ST15-K54 strains causing fatal neonatal sepsis. The presence of pLVPK-associated markers and blaNDM-1 in high-risk clones, and the co-transmission of these genes via conjugation calls for surveillance of these strains. IMPORTANCE Klebsiella pneumoniae is a leading cause of sepsis in newborns and adults. Among the 2 major pathotypes of K. pneumoniae, classical (cKP) and hypervirulent (hvKP), hvKP causes community-acquired severe fatal invasive infections in even healthy individuals, as it possesses several virulence factors. The lack of comprehensive studies on neonatal septicemic hvKPs prompted this work. Nearly 26% diverse hvKP strains were recovered possessing several resistance and virulence determinants. The majority of them exhibited strong biofilm-forming and high serum resistance ability. Nine of these strains were also carbapenem (last-resort antibiotic)-resistant, of which 2 high-risk clones (ST11-K2 and ST15-K54) harbored markers (pLVPK) noted for their virulence, and were lethal in the mouse model. Genome-level characterization of the high-risk clones showed resemblance with the other hvKP reference genomes. The presence of transmissible carbapenem-resistant gene, blaNDM, along with pLVPK-markers calls for vigilance, as most clinical microbiology laboratories do not test for them.

Clinical and Microbiological Characteristics of Endogenous Klebsiella pneumoniae Endophthalmitis in a Tertiary Hospital in Shanghai, China

ABSTRACT Purpose To investigate the clinical features of endogenous Klebsiella pneumoniae endophthalmitis (EKPE) and the molecular characteristics of pathogenic K. pneumoniae. Methods A retrospective study was conducted between January 2014 and January 2021. Clinical data were extracted. K. pneumoniae isolates cultured from EKPE patients’ specimens were characterized by antimicrobial susceptibility testing, multilocus sequence typing, capsular serotyping, and virulence gene profiles. Virulence phenotypes were confirmed by mouse lethality assay. Results Thirty-nine patients (47 eyes) were diagnosed with EKPE. Administration of systemic antibiotics and eye treatment within 24 hours of ocular symptom onset was statistically associated with better visual outcomes (p = .007). Sixteen K. pneumoniae isolates were collected, three of which were carbapenem-resistant and avirulent. Only salmochelin encoding gene was harbored by all isolates. Conclusions Systemic and eye treatment administered within 24 hours from the onset improves visual prognosis. Besides hypervirulent strains, avirulent carbapenem-resistant K. pneumoniae can cause EKPE.

Niclosamide Modulates the Cellular and Humoral Immune Response in Balb/c Mice.

Niclosamide, a STAT3 inhibitor, is widely under investigation due to its anti-cancer properties. STAT3 also exhibits an exciting role in the immune responses. This study aimed to evaluate the impact of niclosamide on immune response of mice. Niclosamide was administered to balb/c mice. To evaluate cell-mediated immune response, a contact-hypersensitivity (CHS) test, cyclophosphamide-induced neutropenic assay, and carbon clearance test were performed, whereas a humoral immune response was evaluated by hemagglutination assay (HA) and mice lethality test. The concentration of TGF-β1 was determined by enzyme-linked immunosorbent assay (ELISA) on murine peritoneal macrophages. In the CHS test, niclosamide caused a decrease in skin thickness, significantly exhibiting a decrease in inflammation. A highly significant decrease in overall leukocyte count (lymphocytes and neutrophils) was observed before and after cyclophosphamide injection as compared with the control group. However, only a highly significant decrease in the neutrophil percentage was observed. Niclosamide has decreased the phagocytic process immensely compared with the control. In the HA titer, niclosamide was found to reduce the antibodies' titer compared with the negative control group. In the mice lethality test, the treatment groups have shown an increase in the percentage of mortality. TGF-β1 elevated in peritoneal macrophages when treated with niclosamide, in a dose-dependent manner. Niclosamide exerts potent immunomodulatory effects by significantly suppressing cell-mediated and humoral immune responses and increasing the levels of TGF-β1 in mice. Niclosamide might be added as an adjuvant to immunosuppressive drugs for the treatment of autoimmune diseases.

Botulinum toxin antibody titres: measurement, interpretation, and practical recommendations

Botulinum toxin (BT) therapy may be blocked by antibodies (BT-AB) resulting in BT-AB induced therapy failure (ABF). BT-AB may be detected by the mouse lethality assay (MLA), the mouse diaphragm assay (MDA) and the sternocleidomastoid test (SCMT). For the first time, we wanted to compare all three BT-AB tests and correlate them to subjective complaint of complete or partial secondary therapy failure in 37 patients with cervical dystonia (25 females, 12 males, age 51.2 ± 11.4 years, disease duration 12.4 ± 6.3 years). Complaint of therapy failure was not correlated with any of the BT-AB tests. MDA and MLA are closely correlated, indicating that the MDA might replace the MLA as the current gold standard for BT-AB measurement. The SCMT is closely correlated with MDA and MLA confirming that BT-AB titres and BT's paretic effect are in a functional balance: low BT-AB titres are reducing BT's paretic effect only marginally, whereas high BT-AB titres may completely block it. When therapy failure is classified as secondary and permanent, BT-AB evaluation is recommended and any BT-AB test may be applied. For MDA > 10 mU/ml, MLA > 3 and SCMT < 25%, ABF is highly likely. MDA < 0.6 mU/ml are therapeutically irrelevant. They are neither correlated with pathologic MLA nor with pathologic SCMT. They should not be the basis for treatment decisions, such as switching dystonia therapy to deep brain stimulation. All other results are intermediate results. Their interactions with therapy efficacy is unpredictable. In these cases, BT-AB tests should be repeated or one or two additional test methods should be applied.

Phenotypic characterization, detection of virulence factors, and antibacterial susceptibility profile of clinical isolates of Klebsiella pneumoniae

Klebsiella pneumoniae has been one of the most occurring bacteria in clinical samples causing community-acquired and nosocomial infections. It has been developing resistance to most antimicrobial agents. These have increased the pathogenicity and the chance for the evolvement of more invasive K. pneumoniae. The study was aimed at the identification and detection of Klebsiella pneumoniae from clinical samples. Some selected virulence factors were detected and the antibacterial susceptibility profile of the isolates was conducted. The incubation period, infective dose, and mortality rate of K.pneumoniae were measured using Baggs albino rats (BALB/c) strain. The isolation and identification of 56 Klebsiella pneumoniae from 223 clinical samples were made. Out of 56 K. pneumoniae isolates, all manifested mucoid phenotype, 44(78.6%) capsule antigen and 13(23.2%) Siderophore. The antimicrobial test conducted identified Zinacef 51(91.1%), Rocephin 50 (89.3%), and Ampiclox 50 (91.1%) as the most resistant. Ciprofloxacin 49 (87.5%), chloramphenicol 35(62.5%), Gentamycin 32(57.1%), and Amoxicillin 28 (50%) were the most susceptible to Klebsiella pneumoniae. The mouse lethality test shows that K. pneumoniae hypermucoviscous strain can cause 41.7% lethality and 66.7% mortality in Baggs Albino rats. Meanwhile an infection dose 105cfu/ml, 107cfu/ml and 109cfu/ml produced an incubation period of 9 days, 7days and 5days. The chi-square test shows no significant association between the isolate and the gender of the patients, but there is a significant association between the samples and isolates identified p &lt;0.05. Likewise, the association between mucoid phenotype, capsule antigen, siderophore, and the type of infection is significant (p&lt;0.05). It is finally concluded that K. pneumoniae is the second most prevailing bacterium causing community-acquired infection the resistant pattern recorded identified the test bacteria as a multidrug-resistant strain and manifestation of the virulent factor depends on the type and site of infection caused by the K. pneumoniae.

Xenografts on nude mouse diaphragm of human DU145 prostate carcinoma cells: mesothelium removal by outgrowths and angiogenesis

ABSTRACT Human prostate carcinoma DU145 cells, androgen-independent malignant cells, implanted in the athymic nu/nu male mouse, developed numerous tumors on peritoneal and retro-peritoneal organs whose growth aspects and vascular supply have yet to be investigated with fine structure techniques. A series of necropsies from moribund implanted mice diaphragms were examined with light, scanning, and transmission electron microscopy. DU145 xenografts installations, far away from the implanted site, were described as the smallest installation to large diaphragm outgrowths in moribund mice. Carcinomas did not show extracellular matrix and, reaching more than 0.15 mm in thickness, they revealed new structures in these outgrowths. Voids to be gland-like structures with mediocre secretion and, unexpectedly, intercellular spaces connected with fascicles of elongated DU145 cells that merged with a vascular supply originated from either the tumor cells and/or some perimysium vessels. In the largest carcinomas, most important vascular invasions coincidently accompanied the mouse lethality, similarly to human cancers. This androgen-independent model would be useful to study tumor outgrowth’s changes related to testing anticancer strategy, including anti-angiogenic therapies involving toxicity, simultaneously with those of other vital organs with combined biomolecular and fine structure techniques.

Genetic characterization and passage instability of a novel hybrid virulence plasmid in a ST23 hypervirulent Klebsiella pneumoniae.

Hypervirulent variants of Klebsiella pnuemoniae (hvKP), which causes life-threatening infections, is a global priority pathogen and frequently harbours virulence plasmids. The virulence plasmids have emerged as the predominant vehicles carrying the major pathogenic determinants of hypermucoviscosity and hypervirulence phenotypes. In the present study, we characterized a novel virulence plasmid in AP8555, an ST23 hvKP strain, which induced a metastatic infection and fatal septic shock in a critically ill patient. The serum killing assay, the quantitative biofilm formation assay, the G.mellonella infection model, and the mouse lethality assay demonstrated that AP8555 was almost as virulent as the hvKP strain NUTH-K2044. The plasmid pAP855 could be conjugated to Klebsiella quasipneumoniae ATCC700603 and E. coli J53 at a frequency of 7.2× 10−5 and 8.7× 10−7, respectively. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHI1B/IncFIB/IncFII and presented high similarity to the pK2044 plasmid. In contrast, a 130-kb large-fragment insertion was observed on the plasmid, which introduced a genetic hybrid zone with multiple conjugation-related genes of type IV secretion systems (T4SS) and CcdAB toxin-antitoxin systems (TAS) to the plasmid. In the transconjugants, the presence of pAP855 had a negative impact on bacterial fitness, but enhancing the virulence-associated phenotypes. In vitro evolution experiments showed that pAP855 in the transconjugants could not be stably inherited after 10 days of passage. Our study not only reports a novel hybrid plasmid but also highlights the putative pathway of conjugative virulence plasmid formation and evolution by means of genetic rearrangement through sequence insertion. These findings indicate that structural versatility could contribute to the dissemination of cointegrate virulence plasmid, although the plasmid incurred a fitness cost. Therefore, continuous monitoring the acquisition of conjugative virulence plasmids may have critical value for plasmid research and increase awareness of hvKP.

The Anti-Virulence Effect of Sub-Minimal Inhibitory Concentrations of Levofloxacin on Hypervirulent Klebsiella pneumoniae.

BackgroundHypervirulent Klebsiella pneumoniae (hvKp) is best described as a virulent pathogen and generally associated with the hypermucoviscosity phenotype. Increased capsule and aerobactin production are established important hvKp-specific virulence factors. Although hvKp strains have been relatively susceptible to antimicrobials, given the high morbidity and mortality, there is a critical need for alternative strategies for the treatment of hvKp infections. Thus, the anti-virulence therapy has been targeted for the hvKp development of therapeutics.Materials and MethodsFour hvKp isolates with hypermucoviscous phenotype were used in our experiments. Mucoviscosity of the capsule can be assessed by low-speed centrifugation of cultures. CPS amount was determined by glucuronic acid content. The capsule thickness was measured under microscope after ink staining. The transcriptions of gene were measured by quantitative real-time PCR (qRT-PCR). The effect of levofloxacin on the resistance of K. pneumoniae to phagocytosis by macrophages and mouse lethality assay was observed.ResultsOur data revealed that sub-Minimal Inhibitory Concentrations (sub-MIC) of LVX reduce mucoviscosity and CPS production of hvKp. Microscopic observations demonstrated that the capsule of hvkp bacteria became thinned after treatment with LVX. qRT-PCR showed decreased transcript levels of rmpA, wzi, magA, iroN and icuA genes. Down-regulation of these virulence genes occurred leading to increased susceptibility to phagocytosis by macrophages. Mouse lethality assay revealed that the wild strain had the LD50 of 103 CFU, while the sub-MIC LVX-treated bacteria had the LD50 of 105 CFU.ConclusionOur data suggested that LVX may serve as a potential anti-virulence agent for refractory infection by hvKp.