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Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization.

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.

Animals↗

Evaluation of potential indicators of viral contamination in shellfish and their applicability to diverse geographical areas.

The distribution of the concentration of potential indicators of fecal viral pollution in shellfish was analyzed under diverse conditions over 18 months in diverse geographical areas. These microorganisms have been evaluated in relation to contamination by human viral pathogens detected in parallel in the analyzed shellfish samples. Thus, significant shellfish-growing areas from diverse countries in the north and south of Europe (Greece, Spain, Sweden, and the United Kingdom) were defined and studied by analyzing different physicochemical parameters in the water and the levels of Escherichia coli, F-specific RNA bacteriophages, and phages infecting Bacteroides fragilis strain RYC2056 in the shellfish produced, before and after depuration treatments. A total of 475 shellfish samples were studied, and the results were statistically analyzed. According to statistical analysis, the presence of human viruses seems to be related to the presence of all potential indicators in the heavily contaminated areas, where E. coli would probably be suitable as a fecal indicator. The F-RNA phages, which are present in higher numbers in Northern Europe, seem to be significantly related to the presence of viral contamination in shellfish, with a very weak predictive value for hepatitis A virus, human adenovirus, and enterovirus and a stronger one for Norwalk-like virus. However, it is important to note that shellfish produced in A or clean B areas can sporadically contain human viruses even in the absence of E. coli or F-RNA phages. The data presented here will be useful in defining microbiological parameters for improving the sanitary control of shellfish consumed raw or barely cooked.

Animals↗

Molecular and phenotypic analyses reveal association of diverse Colletotrichum acutatum groups and a low level of C. gloeosporioides with olive anthracnose.

Anthracnose (Colletotrichum spp.) is an important disease causing major yield losses and poor oil quality in olives. The objectives were to determine the diversity and distribution pattern of Colletotrichum spp. populations prevalent in olives and their relatedness to anthracnose pathogens in other hosts, assess their pathogenic variability and host preference, and develop diagnostic tools. A total of 128 Colletotrichum spp. isolates representing all olive-growing areas in Portugal and a few isolates from other countries were characterized by molecular and phenotypic assays and compared with reference isolates. Arbitrarily primed PCR data, internal transcribed spacer of rRNA gene and beta-tubulin 2 nucleotide sequences, colony characteristics, and benomyl sensitivity showed Colletotrichum acutatum to be dominant (>97%) with limited occurrence of Colletotrichum gloeosporioides (<3%). Among C. acutatum populations, five molecular groups, A2 to A6, were identified. A2 was widely prevalent (89%), coinciding with a high incidence of anthracnose and environmental conditions suitable to disease spread. A4 was dominant in a particular region, while other C. acutatum groups and C. gloeosporioides were sporadic in their occurrence, mostly related to marginal areas of olive cultivation. C. gloeosporioides, isolated from olive fruits with symptoms indistinguishable from those of C. acutatum, showed same virulence rating as the most virulent C. acutatum isolate from group A2. C. acutatum and C. gloeosporioides isolates tested in infected strawberry fruits and strawberry and lupin plants revealed their cross-infection potential. Diagnostic tools were developed from beta-tubulin 2 sequences to enable rapid and reliable pathogen detection and differentiation of C. acutatum groups.

Colletotrichum↗

Potential spectrum of etiological agents of viral enteritis in hospitalized infants.

Fecal specimens were obtained from 1,160 infants and young children with acute nonbacterial gastroenteritis over a period of 2 years. A total of 100 specimens were obtained from age-matched asymptomatic controls. The specimens were examined for the presence of viruses by electron microscopy. Viruses or virus-like particles frequently associated with enteritis were detected in 27% (314 of 1,160) of the symptomatic patients. No viruses or virus-like particles were detected in the 100 control subjects. Rotavirus was detected in 73% (230 of 314) of the virus-positive samples. The mean age of rotavirus-positive patients was 11.5 months, although the patients ranged in age from 2 weeks to 5 years. Of the symptomatic patients, 45 (14%) exhibited small virus-like particles (15 to 40 nm) in the feces in the absence of any other detectable pathogen. Some of the virus-like particles observed in these patients appeared to be similar to astrovirus, and some appeared to be similar to the Otofuke agent or possibly minireovirus. Significantly, however, the mean age of infants with enteritis from whom these small virus-like particles were recovered was 4.5 months (range, 10 days to 19 months). Our findings confirmed the already-known fact that rotaviruses constitute the most important cause of viral enteritis in young children. In addition, small viruses may be an important cause of gastroenteritis in infants under 5 months of age.

Age Factors↗

To culture or not to culture: fecal lactoferrin screening for inflammatory bacterial diarrhea.

Because of its low yield in unselected specimens, stool culture is often cost ineffective. We tested 55 fecal samples from Fairfax Hospital (46 patients with diarrhea and 9 from controls without diarrhea) for lactoferrin by latex agglutination (LFLA) with the Leukotest (Techlab, Blacksburg, Va.) as a marker for inflammatory diarrhea. Of the 28 samples with Salmonella, Shigella, or Campylobacter infection, 93% had detectable fecal lactoferrin at > or = 1:50 (61% had LFLA titers of > or = 1:400), while 83% of 18 samples with rotavirus or no detectable pathogen were LFLA negative at a titer of 1:50 (100% were negative at 1:400). All 9 controls without diarrhea were LFLA negative at 1:50. The use of fecal lactoferrin to screen for inflammatory diarrhea selects specimens for which stool culture is fivefold more likely to yield an invasive bacterial pathogen (reducing the cost per positive result by over $800) and thus may greatly enhance a cost-effective approach to evaluating diarrheal illness.

Adolescent↗

High prevalence of genital mycoplasmas among sexually active young adults with urethritis or cervicitis symptoms in La Crosse, Wisconsin.

Sexually active young adults in the small college town of La Crosse, Wisconsin, were evaluated for conventional sexually transmitted pathogens and tested for infections with mycoplasmas. The prevalence in 65 symptomatic men or women and 137 healthy volunteers (67 men and 70 women) was compared. Urine specimens from both cohorts were tested by ligase chain reaction for Chlamydia trachomatis or Neisseria gonorrhoeae. In addition, the urethral or cervical swabs from the symptomatic subjects were tested by PCR for Mycoplasma genitalium and cultured for Mycoplasma hominis and the ureaplasmas. The results confirmed a relatively low prevalence of gonorrhea among symptomatic men (12%) and chlamydia among symptomatic men (15%) and normal women (3%). In contrast, infections with mycoplasmas, especially the ureaplasmas (57%), were common and the organisms were the only potential sexually transmitted pathogen detected in 40 (62%) symptomatic subjects. Because of the high prevalence, we also evaluated urethral swabs from an additional 25 normal female volunteers and recovered ureaplasmas from 4 (16%) subjects. Additionally, the participants rarely used protection during sexual intercourse and some symptomatic subjects apparently acquired their infections despite using condoms regularly. The findings demonstrate a strong association between abnormal urogenital findings and detection of myoplasmas, particularly ureaplasmas, and suggest the infections will remain common.

Adult↗

Rapid diagnosis of bacterial meningitis by real-time PCR and fluorescence in situ hybridization.

Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.

Cerebrospinal Fluid↗

The lipopolysaccharide-binding protein is a secretory class 1 acute-phase protein whose gene is transcriptionally activated by APRF/STAT/3 and other cytokine-inducible nuclear proteins.

Acute-phase reactants (APRs) are proteins synthesized in the liver following induction by interleukin-1 (IL-1), IL-6, and glucocorticoids, involving transcriptional gene activation. Lipopolysaccharide-binding protein (LBP) is a recently identified hepatic secretory protein potentially involved in the pathogenesis of sepsis, capable of binding the bacterial cell wall product endotoxin and directing it to its cellular receptor, CD14. In order to examine the transcriptional induction mechanisms by which the LBP gene is activated, we have investigated the regulation of expression of its mRNA in vitro and in vivo as well as the organization of 5' upstream regulatory DNA sequences. We show that induction of LBP expression is transcriptionally regulated and is dependent on stimulation with IL-1beta, IL-6, and dexamethasone. By definition, LBP thus has to be viewed as a class 1 acute-phase protein and represents the first APR identified which is capable of detecting pathogenic bacteria. Furthermore, cloning of the LBP promoter revealed the presence of regulatory elements, including the common APR promoter motif APRE/STAT-3 (acute-phase response element/signal transducer and activator of transcription 3). Luciferase reporter gene assays utilizing LBP promoter truncation and point mutation variants indicated that transcriptional activation of the LBP gene required a functional APRE/STAT-3 binding site downstream of the transcription start site, as well as an AP-1 and a C/EBP (CCAAT enhancer-binding protein) binding site. Gel retardation and supershift assays confirmed that upon cytokine stimulation APRF/STAT-3 binds to its recognition site, leading to strong activation of the LBP gene. Unraveling of the mechanism of transcriptional activation of the LBP gene, involving three known transcription factors, may contribute to our understanding of the acute-phase response and the pathophysiology of sepsis and septic shock.

Acute-Phase Proteins↗

Chlamydophila pneumoniae and Mycoplasma pneumoniae in respiratory specimens of children with chronic lung diseases.

BACKGROUND: Persistent infection with Mycoplasma pneumoniae and Chlamydophila pneumoniae has been implicated in the progression or induction of asthma and chronic obstructive pulmonary disease. Evidence for this hypothesis has been obtained in adults either by serological methods or by direct pathogen detection using invasive procedures. METHODS: We investigated nasal brush specimens and induced sputum from 38 children with stable chronic lung disease (asthma, n = 26; chronic bronchitis n = 12) and from 42 healthy controls for the presence of M pneumoniae or C. pneumoniae DNA by polymerase chain reaction (PCR) using nested primers. RESULTS: None of the controls but 23.6% and 10.5% of the children with lung disease had positive PCR for C pneumoniae (p = 0.001) and M pneumoniae (p = 0.044) respectively. Significantly more children with non-atopic asthma than with atopic asthma were positive for C pneumoniae or M pneumoniae (4/8 v 1/18; p = 0.018). There were no unwanted side effects from sputum induction. No correlation was found between detection of Chlamydophila and severity of lung disease. Colonisation with both organisms had occurred before adulthood in a significant proportion of children with stable chronic lung diseases. CONCLUSION: Combining nasal brush specimens with induced sputum may be a useful non-invasive method for studying the role of C pneumoniae and M pneumoniae infection in children with different chronic lung diseases.

Adolescent↗

Diagnostic application of hMLH1 methylation in hereditary non-polyposis colorectal cancer.

Colorectal cancer (CRC) due to mismatch repair (MMR) defect has distinct characteristics among unselected CRCs. These CRCs are biologically less aggressive and, thus, showing better prognosis but less sensitive to the 5FU-based chemotherapy. CRCs with MMR defect derive from both hereditary and sporadic reasons. Germline inactivation of MMR genes (hMLH1, hMSH2, hMSH6, and hPMS2) underlies the hereditary CRC with MMR defect (Lynch syndrome) and epigenetic silencing of hMLH1 gene causes the sporadic CRC with MMR defect. Hereditary and sporadic CRC with MMR defect can be detectable by microsatellite instability (MSI) test or immunohistochemical analysis among general CRCs. Lynch syndrome can be diagnosed by the clinical criteria or by genetic test to detect pathogenic germline mutations in MMR genes. However, both clinical criteria and genetic test are inadequate for the diagnosis of Lynch syndrome. Since genetic test for the diagnosis of the Lynch syndrome is expensive and not always identify pathogenic germline mutations, effective and inexpensive screening program is desirable. Here we propose a possible application of methylation test combined with MSI or pathological analysis as an effective and a cost-saving new strategy for screening of Lynch syndrome.

Adaptor Proteins, Signal Transducing↗

Production of tumor necrosis factor-alpha by blood and lung mononuclear phagocytes from patients with human immunodeficiency virus-related lung disease.

Tumor necrosis factor-alpha (TNF) is a cytokine involved in the pathogenesis of shock and in granuloma formation, tissue necrosis, and fibrosis, in many organ systems, including the lung. It has been suggested that cells from patients infected by the human immunodeficiency virus (HIV + ve) are primed for TNF release. We postulated that TNF release from the alveolar macrophages (AM) of such patients with lung disease might lead to their observed pulmonary dysfunction. We present data confirming that peripheral blood monocytes (PBM) and demonstrating that AM from HIV + ve patients with pulmonary manifestations show significantly greater TNF production than those from HIV-negative (HIV - ve) subjects. In addition, we found sequentially significant increases in TNF production from AM and PBM of HIV + ve patients with no pathogens detected at bronchoscopy (NB), bacterial pneumonia (BP), and those with Pneumocystis carinii pneumonia (PCP). The overall TNF levels were greater from AM than PBM in all groups other than spontaneous production from HIV - ve subjects. Adherent populations of PBM and AM were incubated for 4 h with lipopolysaccharide (10 micrograms/ml) or control medium alone. Cell-free supernatants were examined for the presence of TNF using an immunoassay. The TNF levels (mean +/- SD) in IU/ml from stimulated PBM of the PCP, BP, NB, and control groups, respectively, were 186 +/- 36, 140 +/- 30, 95 +/- 18, and 55 +/- 10 and the spontaneous levels were 123 +/- 25, 100 +/- 22, 75 +/- 24, and 11 +/- 5.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗

Expression of S100A8 correlates with inflammatory lung disease in congenic mice deficient of the cystic fibrosis transmembrane conductance regulator.

BACKGROUND: Lung disease in cystic fibrosis (CF) patients is dominated by chronic inflammation with an early and inappropriate influx of neutrophils causing airway destruction. Congenic C57BL/6 CF mice develop lung inflammatory disease similar to that of patients. In contrast, lungs of congenic BALB/c CF mice remain unaffected. The basis of the neutrophil influx to the airways of CF patients and C57BL/6 mice, and its precipitating factor(s) (spontaneous or infection induced) remains unclear. METHODS: The lungs of 20-day old congenic C57BL/6 (before any overt signs of inflammation) and BALB/c CF mouse lines maintained in sterile environments were investigated for distinctions in the neutrophil chemokines S100A8 and S100A9 by quantitative RT-PCR and RNA in situ hybridization, that were then correlated to neutrophil numbers. RESULTS: The lungs of C57BL/6 CF mice had spontaneous and significant elevation of both neutrophil chemokines S100A8 and S100A9 and a corresponding increase in neutrophils, in the absence of detectable pathogens. In contrast, BALB/c CF mouse lungs maintained under identical conditions, had similar elevations of S100A9 expression and resident neutrophil numbers, but diverged in having normal levels of S100A8. CONCLUSION: The results indicate early and spontaneous lung inflammation in CF mice, whose progression corresponds to increased expression of both S100A8 and S100A9, but not S100A9 alone. Moreover, since both C57BL/6 and BALB/c CF lungs were maintained under identical conditions and had similar elevations in S100A9 and neutrophils, the higher S100A8 expression in the former (or suppression in latter) is a result of secondary genetic influences rather than environment or differential infection.

Animals↗

Detection of a non-pathogenic variant of Anaplasma phagocytophilum in Ixodes ricinus from La Rioja, Spain.

Our aim was to identify variants of Anaplasma phagocytophilum 16S rRNA gene sequences among products amplified from Ixodes ricinus collected in La Rioja, Spain. A. phagocytophilum AP-variant 1, reported as non-pathogenic, was detected in 12 samples (two adults and ten nymphs). This finding could justify the low incidence of human anaplasmosis in our area, despite the high prevalence of A. phagocytophilum in ticks.

Anaplasma phagocytophilum↗

Analysis of the preproPTH gene by denaturing gradient gel electrophoresis in familial isolated hypoparathyroidism.

Familial isolated hypoparathyroidism (FIH) is an inherited metabolic disorder characterized by hypocalcemia and hyperphosphatemia due to deficient secretion of biologically active PTH. We used denaturing gradient gel electrophoresis to screen for mutations in exon 1 and the coding region of the preproPTH gene. Exons 1, 2, and 3 of the preproPTH gene and flanking intronic regions were amplified by polymerase chain reaction using primers that were designed employing the MELT-MAP program. One oligonucleotide from each primer pair was synthesized with a 5'-GC-clamp. Screening of amplified DNA from normal subjects and patients with FIH revealed two single base changes that altered migration of amplified preproPTH gene fragments through denaturing gels: 1) an A----G transition in intron 1; 10 nucleotides upstream of exon 2; and 2) a C----A transversion in exon 3 that conserves the arginine residue at codon 52 (CGA----AGA). By contrast, we did not detect pathogenic mutations in amplified regions of the preproPTH genes of 18 affected members of 5 FIH kindreds. The two polymorphisms occur frequently, and were therefore used to perform linkage analysis in 5 multiplex FIH families. Linkage analysis was inconclusive in 2 families and showed discordance between hypoparathyroidism and any of the preproPTH gene alleles in 2 other families. In another family, analysis was suggestive of linkage between hypoparathyroidism and inheritance of a specific preproPTH gene allele. These results indicate that denaturing gradient gel electrophoresis can be used to identify mutations in defined regions of the preproPTH gene, and to examine linkage of specific preproPTH alleles and inherited disorders of mineral metabolism.

Amino Acid Sequence↗

Lack of Bcl10 gene mutations in laryngeal squamous cell carcinoma.

The Bcl10 gene encodes a protein probably involved in some apoptotic regulatory pathways. Bcl10 mutations lead to the translation of truncated proteins that show gain-of-function transforming activity; it has been suggested that Bcl10 may represent a major target gene for inactivation in many human cancers. To define the frequency of Bcl10 mutations in laryngeal squamous cell carcinoma and their possible association with tumour progression, we investigated a large panel of tumours representative of all grades and stages of malignancy. To detect pathogenic mutations in exons 1, 2 and 3 of the Bcl10 gene, we performed a silver-staining polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis followed by direct DNA sequencing. We revealed the presence of SSCP variants in 18 out of 91 laryngeal tumours. Direct DNA sequencing showed previously described polymorphisms but no pathogenic mutations. We have strong evidence that the Bcl10 gene is not involved in laryngeal carcinogenesis.

Adaptor Proteins, Signal Transducing↗

Genomics for precision surgical source control in anti-microbial resistant infections: A global review with focus on resource-limited settings.

BACKGROUND & OBJECTIVE: Antimicrobial resistance (AMR) critically threatens surgical safety, impairing perioperative prophylaxis and complicating infection management. Timely surgical source control is essential but relies on accurate microbiological diagnosis. Conventional culture-based methods are slow and insensitive, often leading to empirical broad-spectrum therapy. This review evaluates the role of advanced genomic diagnostics in enhancing surgical source control for AMR infections, with a focus on challenges and opportunities in low- and middle-income countries (LMICs) like Pakistan. METHODOLOGY: A narrative review was conducted via a structured search of PubMed, Google Scholar, and ScienceDirect (January 2015-October 2025). Studies involving genomic tools in the management of AMR-related surgical infections were included. Evidence was synthesized thematically, covering genomic platforms, clinical applications, implementation barriers, and LMIC specific perspectives. RESULTS: Genomic tools, particularly metagenomic next-generation sequencing (mNGS) and rapid multiplex PCR, demonstrate superior sensitivity (80.6-95.45%) and faster turnaround times (e.g., roughly 27 hours for mNGS) compared to culture. They improve pathogen detection in complex infections (e.g., prosthetic joints, necrotizing soft tissue), guide targeted antibiotic therapy, and can reduce broad-spectrum use. However, major implementation barriers exist, including high costs, need for specialized infrastructure and expertise, bioinformatic challenges, and ethical data concerns, which are especially pronounced in LMICs. CONCLUSION: Genomic diagnostics offer a powerful approach to accelerate and refine surgical source control in the era of AMR. Strategic investments in local capacity, affordable platforms, and integration with antimicrobial stewardship are needed to realize their potential for improving surgical outcomes, particularly in resource-limited settings.

Antimicrobial resistance↗

Leg ulcer odour detection identifies beta-haemolytic streptococcal infection.

beta-Haemolytic streptococci were identified in bacteriological cultures from 14 of 24 chronic venous leg ulcers in 21 patients. Multi-element odour detection (MEOD) analysis demonstrated a significant difference in odour in those ulcers from which beta-haemolytic streptococci were isolated (p < 0.01). MEOD has potential to detect pathogenic organisms instantaneously in the clinical setting.

Chronic Disease↗

Functional dissection of an innate immune response by a genome-wide RNAi screen.

The innate immune system is ancient and highly conserved. It is the first line of defense and the only recognizable immune system in the vast majority of metazoans. Signaling events that convert pathogen detection into a defense response are central to innate immunity. Drosophila has emerged as an invaluable model organism for studying this regulation. Activation of the NF-kappaB family member Relish by the caspase-8 homolog Dredd is a central, but still poorly understood, signaling module in the response to gram-negative bacteria. To identify the genes contributing to this regulation, we produced double-stranded RNAs corresponding to the conserved genes in the Drosophila genome and used this resource in genome-wide RNA interference screens. We identified numerous inhibitors and activators of immune reporters in a cell culture model. Epistatic interactions and phenotypes defined a hierarchy of gene action and demonstrated that the conserved gene sickie is required for activation of Relish. We also showed that a second gene, defense repressor 1, encodes a product with characteristics of an inhibitor of apoptosis protein that inhibits the Dredd caspase to maintain quiescence of the signaling pathway. Molecular analysis revealed that Defense repressor 1 is upregulated by Dredd in a feedback loop. We propose that interruption of this feedback loop contributes to signal transduction.

Amino Acid Sequence↗