- Volume 67, Issue 9, 2018
Volume 67, Issue 9, 2018
- Review
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Beyond proteostasis: Roles of type I chaperonins in bacterial pathogenesis
More LessNearly all bacterial species express two or more chaperonin genes. Recent data indicate that type I chaperonins may be key players in bacterial infections. This is partly due to the well-known contribution of chaperonins in cellular proteostasis, the latter being compromised during bacterial host infection. In addition to their protein-folding activity, it has been revealed that certain chaperonins also exhibit moonlighting functions that can contribute in different ways to bacterial pathogenicity. Examples range from inducing adhesion molecules in Chlamydophila pneumoniae to supporting intracellular survival in Mycobacterium tuberculosis and Leishmania donovani, to inducing cytokines in Helicobacter pylori to promoting antimicrobial resistance in Escherichia coli, amongst others. This article provides a thorough reviews of our current understanding of the different mechanisms involving type I chaperonins during bacteria–host interactions, and suggests new areas to be explored and the potential of finding new targets for fighting bacterial infections.
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- Antimicrobial Resistance
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Pyrosequencing: a rapid and effective sequencing method to diagnose drug-resistant tuberculosis
Purpose. This study was undertaken to evaluate the efficiency of the pyrosequencing (PSQ) assay for the rapid detection of resistance to rifampicin (RIF), fluoroquinolones (FQs) and second-line injectables (SLIs) such as capreomycin (CAP) and kanamycin (KAN) in Mycobacterium tuberculosis (Mtb) clinical isolates.
Methodology. Pyrosequencing is a simple and accurate short read DNA sequencing method for genome analysis. DNA extraction from Mtb clinical isolates was performed using Tris-HCl buffer and chloroform. The rpoB (RIF), gyrA (FQs) and rrs (aminoglycosides) genes were amplified, followed by sequencing using the PyroMark Q24 ID system. The PSQ results were compared with the results from the conventional drug susceptibility testing performed in the laboratory.
Results. The sensitivity of the PSQ assay for the detection of resistance to RIF, FQ, CAP and KAN was 100 %, 100 %, 40 % and 50 %, respectively. The specificity of the PSQ assay was 100 %.
Conclusion. The PSQ assay is a rapid and effective method for detecting drug resistance mutations from Mtb clinical isolates in a short period of time.
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In vitro activity of β-lactams in combination with avibactam against multidrug-resistant Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Achromobacter xylosoxidans isolates from patients with cystic fibrosis
More LessThe in vitro activity of anti-pseudomonal β-lactams in combination with avibactam was evaluated against 54 multidrug-resistant non-fermenting Gram-negative bacilli isolated from cystic fibrosis patients. Avibactam increased and/or restored the antibacterial activities of ceftazidime and aztreonam against Pseudomonas aeruginosa and Stenotrophomonas maltophilia, respectively. No β-lactam–avibactam combination was active against Achromobacter xylosoxidans.
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Genomic reorganization by IS26 in a bla NDM-5-bearing FII plasmid of Klebsiella pneumoniae isolated from a patient in Japan
An NDM-5-producing Klebsiella pneumoniae ST147 strain was isolated from a Japanese patient who had not travelled abroad in at least 5 years. Whole-genome sequencing revealed a genomic rearrangement in an FII plasmid harbouring bla NDM-5 due to the replicative transposition of IS26. A hypothetical structure was proposed for its ancestral plasmid, and comparative genomic analysis of the plasmid suggested the dissemination of structurally similar plasmids harbouring bla NDM-5 in Asian and Middle Eastern countries.
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- Clinical Microbiology
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Quantification of major constituents of biofilms in occluded pancreatic stents
More LessPurpose. Biofilms comprise bacterial populations enclosed in a matrix that attaches to surfaces such as medical stents. We characterized the biofilm components in occluding pancreatic stents and investigated potential factors for the formation of the biofilms.
Methodology. The clinical details of 24 patients (M : F, 15 : 9) undergoing pancreatic stent retrieval were noted and the retrieved stents were processed for the quantification of biofilm proteins and polysaccharides and the molecular identification of bacteria.
Results. The patients’ ages ranged from 16 to 62 years. The underlying indications for stent insertion were bile duct stone prophylaxis against post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (n=7; 29.1 %) and pancreatic ductal leaks (n=17; 70.9 %). The retrieved stent sizes were 5 Fr (n=5; 20.8 %) and 7 Fr (n=19; 79.2 %), with a mean insertion duration of 103 days. The polybacteria detected by PCR in 95.8 % of the stents were Pseudomonas (n=8), Staphylococcus (n=8), Serratia (n=5), Aeromonas (n=4), Proteus (n=4), Klebsiella (n=4), Escherichia coli (n=4), Enterococcus (n=4), Streptococcus (n=4), Citrobacter (n=3), Bacillus (n=2), Enterobacter (n=1), Vibrio (n=1) and Clostridium (n=1). Several other organisms were identified by sequencing. The mean protein concentration was 0.585±0.29 mg ml−1 and the mean polysaccharide concentration was 0.054±0.03 mg ml−1. No significant differences were observed in the quantity of proteins and polysaccharides (P=0.933) for various factors, namely gender, presence of cholangitis, indications for stenting, stent sizes and duration of indwelling stents. Age was found to be a significant (P=0.013) factor for protein deposition for those aged >50 years.
Conclusion. The majority of the pancreatic stents grew polymicro-organisms, and those from patients aged >50 years showed significant deposition of protein, which is a key element in biofilm formation. Understanding the constituents of the biofilms in pancreatic stents could be very useful in developing future strategies for the prevention of biofilm formation.
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Mycoplasma pneumoniae DNA detection and specific antibody class response in patients from two tertiary care hospitals in tropical Sri Lanka
More LessPurpose. Respiratory tract infections are a major cause of global morbidity and mortality. Pneumonia is the ninth leading cause of mortality in Sri Lanka. Atypical pathogens cause about one-fifth of community-acquired pneumonia, while Mycoplasma pneumoniae accounts for about 50 %. This study aimed to determine the seroprevalence of M. pneumoniae respiratory tract infections in Sri Lanka while attempting to understand the relationships between the serology and PCR.
Methodology. Paired sera from 418 adult patients (pneumonia, n=97; bronchitis, n=183; pharyngitis, n=138) and 87 healthy controls were studied. IgM, IgG and IgA antibodies were tested by M. pneumoniae enzyme-linked immunosorbent assay (ELISA). Positive IgM and or IgG seroconversion was considered to be seropositive. M. pneumoniae DNA were tested by PCR in age and gender-matched seropositives and seronegatives.
Results. M. pneumoniae IgG was in 14.4 % (14/97), 6.0 % (11/183) and 1.5 % (2/138) of pneumonia, bronchitis and pharyngitis patients, respectively, whilst IgM was in 6.2 % (6/97), 1.1 % (2/183) and 0 % (0/138), respectively. Amongst the pneumonia seropositives, 64.7 % (11/17) showed IgG alone, 17.5 % (3/17) showed IgM alone and 17.5 % (3/17) showed IgM and IgG. Amongst the bronchitis seropositives, 84.6 % (11/13) had IgG alone and 15.4 % (2/13) had IgM alone. In the pharyngitis seropositives, only IgG was detected 100 % (2/2). M. pneumoniae DNA was in 52.2 % (12/23) of seropositives and 15.4 % (4/26) of seronegatives. In pneumonia or bronchitis patients, specific DNA was in 77.8 % (7/10) and 50 % (6/12) of patients, respectively. M. pneumoniae DNA was not found in pharyngitis patients. Of the seropositive PCR-negative pneumonia patients, 66.7 % (2/3) showed IgG alone and 33.3 % (1/3)showed IgM alone. In bronchitis patients, 83.3 % (5/6) showed IgG alone and 16.7 % (1/6) showed IgM alone. Of the seronegative PCR-positive patients, 16.7 % (2/12) had pneumonia and 18.2 % (2/11) had bronchitis.
Conclusion.The serological evidence for M. pneumoniae infection in Sri Lanka comprised the following prevalences: 17.5 % (17/97), 7.1 % (13/183) and 1.4 % (2/138) in adults with pneumonia, bronchitis or pharyngitis, respectively. M. pneumoniae DNA was in 52.2 % (12/23) of seropositives and 15.4 % (4/26) of seronegatives. IgG was predominant in PCR positives and negatives.
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High rates of Mycobacterium fortuitum isolation in respiratory samples from Iranian patients with suspected tuberculosis: is it clinically important?
Purpose. Although Mycobacterium fortuitum (M. fortuitum) is not an organism rarely isolated from respiratory samples, its clinical importance is still not fully understood, which therefore prompted our current study.
Methodology. We evaluated respiratory samples from 6800 patients with suspected tuberculosis from May 2014 to May 2016, for the detection of M. fortuitum using phenotypic and genotyping methods.
Results/Key findings. Of the 40 patients with M. fortuitum lung disease, 35 had two or more positive culture results. The mean age of these 35 patients was 50.7±18.4 years, and 20 (57.1 %) were men. Sputum (68.6 %), haemoptysis (51.4 %), cough (45.7 %) and gastroesophageal disease (22.9 %) were the major presenting symptoms. Cystic fibrosis, other bacterial lung diseases and lung cancer were the main underlying pulmonary diseases. Five patients (12.5 %) were human immunodeficiency virus (HIV) positive. The most common chest X-ray findings were reticulonodular opacities (53.3 %). Multivariate logistic regression analysis revealed that cigarette smoking history (OR 0.334, 95 % CI 0.125–0.843, P=0.048) and underlying lung disease (OR 0.393, 95 % CI 0.216–0.588, P=0.023) were significant predictors for positive M. fortuitum infection.
Conclusion. These results demonstrated the high frequency of M. fortuitum in respiratory samples and that this bacterium causes transient infection or colonization in patients with underlying pulmonary conditions, such as cystic fibrosis and cigarette smoking-induced. Additionally, it appears that infection with M. fortuitum is particularly common and may be important in patients with HIV.
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Isolation and characterization of Leptospira interrogans from two patients with leptospirosis in Western Province, Sri Lanka
Leptospirosis is an endemic infectious disease causing considerable morbidity and mortality in Sri Lanka; however, reports on the isolation of Leptospira from infected patients in Sri Lanka have been largely unavailable since the 1970s. Two isolates were obtained and characterized from 100 blood cultures from leptospirosis-suspected patients. Phylogenic analysis of partial flaB gene sequences identified the isolates as Leptospira interrogans. The patient serum samples from which Leptospira was isolated reacted with the Leptospira serogroups Sejroe and Canicola at a titre of 1 : 200. Exposure to domestic sewage and gutters filled with muddy water was suspected to be the source of infection in these two culture-positive patients. This study reports the successful isolation of pathogenic Leptospira from two patients in Western Province, Sri Lanka.
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Evaluation of the FilmArray Blood Culture Identification Panel compared to direct MALDI-TOF MS identification for rapid identification of pathogens
More LessTo improve time to identification of pathogens and detection of resistance genes, we evaluated the BioFire FilmArray Blood Culture Identification Panel (BCID) as compared to: (1) direct MALDI-TOF MS (DM) and (2) standardized culture-based identification (ID) with antibiotic susceptibility testing (AST). BCID gave an accurate identification in 102/112 (91 %) of cases (102/103 for on-panel organisms). DM gave an accurate identification in 91/112 (81 %) of cases, with 13/91 (14 %) requiring repeat testing from the residual pellet. The mean time to an identification result was 2.4 and 2.9 h for BCID and DM, respectively. Standardized ID and AST results were available at a mean time of 26.5 and 33 h, respectively. There were 44 BCID/DM results that had an antimicrobial treatment change made based on rapid identification and resistant gene detection of pathogens. Both BCID and DM are accurate and rapid methods for the identification of new positive blood culture pathogens.
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- Disease, Diagnosis and Diagnostics
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Novel multiplex PCR assay for identification and subtyping of enteroinvasive Escherichia coli and differentiation from Shigella based on target genes selected by comparative genomics
More LessPurpose. Both Shigella and enteroinvasive Escherichia coli (EIEC) can cause enterocolitis, but they have a distinct epidemiology and public health relevance. Current culture-independent testing (CIT) methods to identify Shigella in faecal samples rely on the ipaH gene as the target, which is also found in EIEC genomes. The aim of this study was to design an assay that can identify EIEC in cultures from CIT ipaH-positive samples.
Methodology. Shigella and EIEC genomes were screened to find unique regions present in EIEC genomes using a comparative genomics approach and differentiating genetic loci that are suitable PCR targets were identified. The primers for these loci were designed and tested in 6501 and 104 genomes of Shigella and EIEC, respectively.
Results. An assay with two sets of multiplex PCR reactions that differentiates Shigella and EIEC based on the presence/absence of at least two out of six loci was developed and evaluated. The majority of Shigella genomes lacked all six loci, while at least two loci were present in most EIEC genomes. This assay successfully differentiated clinical EIEC from Shigella with a limit of detection of 105 cells ml−1. The sensitivity and specificity were over 95 and 99%, respectively. The assay can further subtype EIEC genomes into their genetic lineages.
Conclusion. This new highly specific assay can assist in the identification of EIEC in ipaH PCR-positive samples and augment the public health laboratory surveillance of EIEC and shigellosis.
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Accuracy of a commercial multiplex PCR for the diagnosis of bacterial vaginosis
More LessPurpose. Bacterial vaginosis (BV) is a common clinical condition characterized by odorous vaginal discharge, vaginal itching and/or burning. BV can occur when vaginal lactobacilli are depleted and replaced by diverse anaerobic bacteria. We evaluated a commercial multiplex PCR (ATRiDA) for the diagnosis of BV.
Methods. Cervicovaginal samples were included from women reporting urogenital symptoms and from women notified for sexually transmitted infections (STI) – who were not (necessarily) symptomatic. Clinical BV diagnoses were obtained from electronic patient files. The ATRiDA test measures the loads of Gardnerella vaginalis, Atopobium vaginae and Lactobacillus species in relation to overall bacterial load. The ATRiDA test outcome was compared to the clinical BV diagnosis and to vaginal microbiota composition, determined by 16SrRNA gene sequencing.
Results. We included samples from 185 women reporting urogenital symptoms, of whom 81 had BV and 93 women who were notified for an STI, of whom 16 had BV. Overall, compared to the clinical BV diagnosis, the ATRiDA test demonstrated high sensitivity (96.9 %) and moderate specificity (70.2 %). The negative predictive value was high (>97.3). The positive predictive value differed by study group and was highest in women reporting urogenital symptoms (78.2 %). Sequencing showed that 54 % of women who had an ATRiDA BV-positive test outcome, but who were not clinically diagnosed with BV, had diverse anaerobic vaginal microbiota (asymptomatic vaginal dysbiosis).
Conclusion. The ATRiDA test is a sensitive method for the detection of BV but, given the high occurrence of asymptomatic vaginal dysbiosis, a positive test outcome should be interpreted together with clinical symptoms.
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Development of a species-specific PCR-RFLP targeting rpoD gene fragment for discrimination of Aeromonas species
Purpose. The taxonomy of Aeromonas keeps expanding and their identification remains problematic due to their phenotypic and genotypic heterogeneity. In this study, we aimed to develop a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism assay targeting the rpoD gene to enable the differentiation of aeromonads into 27 distinct species using microfluidic capillary electrophoresis.
Methodology. A pair of degenerate primers (Aero F: 5′-YGARATCGAYATCGCCAARCGB-3′ and Aero R: 5′-GRCCDATGCTCATRCGRCGGTT-3′) was designed that amplified the rpoD gene of 27 Aeromonas species. Subsequently, in silico analysis enabled the differentiation of 25 species using the single restriction endonuclease AluI, while 2 species, A. sanarelli and A. taiwanensis, required an additional restriction endonuclease, HpyCH4IV. Twelve type strains (A. hydrophila ATCC7966T, A. caviae ATCC15468T, A. veronii ATCC9071T, A. media DSM4881T, A. allosaccharophila DSM11576T, A. dhakensis DSM17689T, A. enteropelogens DSM7312T, A. jandaei DSM7311T, A. rivuli DSM22539T, A. salmonicida ATCC33658T, A. taiwanensis DSM24096T and A. sanarelli DSM24094T) were randomly selected from the 27 Aeromonas species for experimental validation.
Results/key findings. The twelve type strains demonstrated distinctive RFLP patterns and supported the in silico digestion. Subsequently, 60 clinical and environmental strains from our collection, comprising nine Aeromonas species, were used for screening examinations, and the results were in agreement.
Conclusion. This method provides an alternative method for laboratory identification, surveillance and epidemiological investigations of clinical and environmental specimens.
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Comparison of the clinical performance of OmniPlex-HPV and GeneFinder HPV for the detection and genotyping of human papillomaviruses in cervical specimens
Purpose. Human papillomavirus (HPV) infection in women is known to promote the development of cervical neoplasia. Specific HPV genotypes are more highly associated with disease, and therefore detection and genotyping of HPV infection is critical for preventing and effectively treating cervical cancer. Consequently, various assays using diverse technologies have been developed to detect HPV genotype. Recently the OmniPlex-HPV and GeneFinder HPV methods, based on PCR and Luminex xMAP liquid bead microarray technologies, were developed for the detection of 40 and 32 HPV genotypes, respectively. The purpose of this study was to compare the clinical performance of OmniPlex-HPV and GeneFinder HPV.
Methodology. The study included 300 cytology-confirmed cervical swab specimens. In cases where there was a discrepancy between the two assay results, type-specific direct sequencing was performed.
Result. We found a high overall agreement between OmniPlex-HPV and GeneFinder HPV for detecting the presence or absence of high-risk HPV (HR HPV) (90.7 %, κ=0.810). However, OmniPlex-HPV showed greater sensitivity than GeneFinder HPV in the identification of multiple genotype-infected samples. Specifically, diagnostic sensitivities for HR HPV positivity in high-grade squamous intra-epithelial lesions (HSIL) were 100.0 % for OmniPlex-HPV and 96.8 % for GeneFinder HPV.
Conclusions. Our results suggest that OmniPlex-HPV and GeneFinder HPV are highly comparable for the detection and genotyping of HPV, but OmniPlex-HPV displays greater accuracy in cases of multiple HPV infection.
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Multi-centric validation of an in-house-developed beacon-based PCR diagnostic assay kit for Chlamydia and Neisseria and portable fluorescence detector
Objective. The development of an accurate, sensitive, specific, rapid, reproducible, stable-at-room-temperature and cost-effective diagnostic kit, and a low-cost portable fluorescence detector to fulfil the requirements of diagnostic facilities in developing countries.
Methods. We developed the ‘Chlamy and Ness CT/NG kit’ based on molecular beacons for the detection of Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG). Multi-centric evaluation of the CT/NG kit was performed using the commercially available nucleic acid amplification test (NAAT)-based FTD Urethritis basic kit for comparison from December 2014 to November 2016. The stability of the kit reagents at 4 and 37 ˚C and the inter-day reproducibility of results were also analysed.
Results. The sensitivity and specificity of the kit were found to be 95.83 and 100.00 % for the detection of C. trachomatis and 93.24 and 99.75 % for N. gonorrhoeae, respectively, when tested against the commercial kit. The positive predictive value (PPV) was 100.00 and 98.57 %, whereas the negative predictive value (NPV) was 99.54 and 98.79 % for C. trachomatis and N. gonorrhoeae, respectively. Analysis of the kappa statistics enhanced the ‘inter-rater’ κ=0.976 for Chlamydia and κ=0.943 for Neisseria.
Conclusion. Our kit was found to be as sensitive and specific as commercially available kits. Its low cost and ease of use will make it suitable for the routine diagnosis of C. trachomatis and N. gonorrhoeae in the resource-limited settings of developing countries.
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Multicenter assessment of the rapid Unyvero Blood Culture molecular assay
Purpose. Bloodstream infections remain an important cause of morbidity and mortality. Rapid diagnosis can reduce the time from empiric antimicrobial therapy to targeted therapy and improve patient outcomes.
Methodology. The fully automated Unyvero Blood Culture (BCU) Application (Curetis GmbH) can identify a broad panel of pathogens (36 analytes covering over 50 pathogens) and 16 antibiotic resistance gene markers simultaneously in about 5 h. The assay was evaluated in three clinical laboratories in comparison to routine microbiological procedures.
Results. A total of 207 blood cultures were included in the study, and 90.5 % of the species identified by culture were covered by the Unyvero BCU panel with an overall sensitivity of 96.8 % and specificity of 99.8 %. The time to result was reduced on average by about 34 h. The assay accurately identified 95 % of the species, including 158/164 monomicrobial and 7/9 polymicrobial cultures. The Unyvero BCU Cartridge detected a large number of resistance markers including mecA (n=57), aac(6′)aph(2′′) (n=40), one vanB resistance gene, and six instances of bla CTX-M.
Conclusion. The Unyvero BCU Application provided fast, reliable results, while significantly improving turnaround time in blood culture diagnostics.
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Performance evaluation of five commercial assays in assessing seroprevalence of HEV antibodies among blood donors
Introduction. Although hepatitis E virus (HEV) is mainly transmitted via the faecal–oral route, the rate of HEV transmission via blood donation is on the rise. However, the seroprevalence of HEV among blood donors is not well established and is thought to be affected by the type of diagnostic assay used. We aimed to evaluate performance and correlation among widely used commercial diagnostic assays for the seroprevalence assessment of HEV-IgM/IgG among blood donors.
Methodology. A total of 1049 blood donor samples were tested for HEV IgG and IgM using different enzyme immunoassays (Wantai, Eruoimmune, MP diagnostics, Mikrogen immunoblot, HEV-IgM rapid test). The performance of each assay was evaluated according to our established silver standard value based on three or more IgG concordant assay results.
Results. HEV seroprevalence varied considerably using these assays, ranging from 10.1 % (Euroimmune-ELISA) to 18.0 % (Wanti-ELISA) for HEV-IgG, and from 0.2 % (Wanti-ELISA) to 2.6 % (MP Rapid test) for HEV-IgM. A total of 155 of 216 (71.6%) samples tested positive for HEV-IgG by three or more concordant assays. On the other hand, IgM assays showed poor agreement as only 7.6 % (4/52) of the specimens were positive according to three or more concordant assay test results. All HEV-IgG assays revealed high sensitivity and specificity (ranging 96.5–100 %),and excellent Kappa concordance (0.88–0.95), except for Euroimmun ELISA (sensitivity=61.5 %, kappa=0.63). MP ELISA showed the highest levels of sensitivity (100 %) and specificity (98.5 %).
Conclusions. Due to discrepancies in the performance of various IgG and IgM assays, seroprevalence studies should be based on furher confirmatory testing for decisive conclusions to be reached.
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Genotyping based on thermal denaturation of amplification products identifies species of the Mycobacterium tuberculosis complex
More LessPurpose. To develop a fast and inexpensive genotyping assay to identify the Mycobacterium tuberculosis complex (MTC) species most prevalent in human tuberculosis (TB), based on the thermal denaturation profiles of PCR products from mycobacterial 16S rDNA and three MTC genomic regions of difference (RD).
Methodology. Genotypes were determined by the presence and thermal denaturation profiles of the amplicons generated in the ‘preliminary’ PCR mixture (16S rDNA), followed by those of the simultaneous D1 (RD9+, RD1−) and D2 (RD4+, RD4−) PCR mixtures. The 16S rDNA profile identifies the genus Mycobacterium; the absence of any additional RD profile identifies Mycobacterium non-tuberculous (MNT) strains; additional RD4+ and RD9+ profiles without RD1− identify M. tuberculosis; an additional RD4+ profile per se identifies M. africanum; an additional RD4− profile per se identifies Mycobaterium bovis; additional RD1− and RD4− profiles identify M. bovis BCG.
Results. Genotypes of a panel with 44 mycobacterial strains coincided in 16 MB and five non-MTC strains; in the remaining 23 MTC strains, 17 MTB and five MA concordant genotypes and one discordant MB genotype were resolved. The genotypes of 13 human and bovine MTC isolates coincided in all four MB and eight of the nine MTB isolates.
Conclusion. Sensitivity, specificity and positive and negative predictive values of the method are 100 % for the genus Mycobacterium, which resolves MB, MTB and MA genotypes. Species/genotype agreement is 97.7 % for the panel and 92.3 % for the MTC isolates. This method may be advantageously used to identify the most prevalent MTC species in humans.
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Effect of storage time and temperature on the detection of Pseudomonas aeruginosa, Acanthamoeba and Herpes Simplex Virus from corneal impression membranes
More LessThe effect of storage time and temperature on the recovery of pathogen DNA from polytetrafluorethylene (PTFE) was investigated. PTFE impression membranes were inoculated with Pseudomonas aeruginosa, Herpes Simplex Virus-1 (HSV-1) or Acanthamoeba and stored at −70 °C, −20 °C, +4 °C or +35 °C. PCR was performed on days 0, 1, 2, 3, 7 and months 1, 3 and 10 post-inoculation. We found no reduction in the DNA recovery of any of the studied microorganisms for the first 3 days of storage up to +35 °C. For HSV-1 and P. aeruginosa, storage for 3 months at +35 °C was associated with a significant reduction in DNA recovery (P<0.001), but not at +4 °C, −20 °C or −70 °C for 1 month for P. aeruginosa and for 10 months for HSV-1. Acanthamoeba DNA recovery was not affected by any storage parameters (P=0.203). These results will inform the investigation of microbial keratitis where access to microbiological testing is not readily available.
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- Microbial Epidemiology
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Dissemination of newly emerged polymyxin B sensitive Vibrio cholerae O1 containing Haitian-like genetic traits in different parts of India
Purpose. Two natural epidemic biotypes of Vibrio cholerae O1, classical and El Tor, exhibit different patterns of sensitivity against the antimicrobial peptide polymyxin B. This difference in sensitivity has been one of the major markers in biotype classification system for several decades. A recent report regarding the emergence of polymyxin B-sensitive El Tor V. cholerae O1 in Kolkata has motivated us to track the spread of the strains containing this important trait, along with Haitian-like genetic content, in different parts of India.
Methodology. We have collected 260 clinical V. cholerae O1 strains from 12 states in India and screened them for polymyxin B susceptibility. Genetic characterization was also performed to study the tcpA, ctxB and rtxA genotypes by allele-specific polymerase chain reaction (PCR) and nucleotide sequencing.
Results. Interestingly, 88.85 % of the isolates were found to be sensitive to polymyxin B. All of the states, with the exception of Assam, had polymyxin B-sensitive V. cholerae strains and complete replacement with this strain was found in eight of the states. However, from 2016 onwards, all the strains tested showed sensitivity to polymyxin B. Allele-specific PCR and sequencing confirmed that all strains possessed Haitian-like genetic traits.
Conclusion. Polymyxin B-sensitive strains have begun to spread throughout India and may lead to the revision of the biotype classification. The dissemination of these new variant strains needs to be carefully monitored in different endemic populations through active holistic surveillance to understand their clinical and epidemiological consequences.
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Distribution of genes encoding virulence factors and multilocus variable-number tandem-repeat analysis (MLVA) of entero-aggregative Escherichia coli (EAEC) isolated in Iran from patients with diarrhoea
Purpose. Entero-aggregative Escherichia coli (EAEC) is one of the main causes of diarrhoea worldwide. Several virulence factors have been identified in EAEC. This study was conducted to investigate the distribution of virulence factor genes in EAEC strains isolated in Iran from children with diarrhoea, as well as the genetic similarity of these isolates.
Methodology. A total of 37 EAEC isolates were tested for the presence of 11 virulence genes by PCR, and the genetic relatedness of these strains was further determined by multilocus variable-number tandem-repeat analysis (MLVA).
Results. All EAEC isolates were typical EAEC. pic, set1A and set1B were the most prevalent genes, detected in 54.1 % of the isolates, followed by sat (43.2 %), astA (32.4 %), pet (24.3 %), agg4A (24.3 %), sepA (18.9 %), agg3A (13.5 %), sigA (8.1 %), aggA (8.1 %) and aafA (5.4 %). Using MLVA, the 37 isolates were divided into 32 types and classified into five clonal complexes.
Conclusion. This study showed that EAEC is a heterogeneous group of E. coli possessing a broad range of virulence factors. There was no notable association between MLVA patterns and virulence profiles.
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The potential of different molecular biology methods in tracking clones of Acinetobacter baumannii in an ICU setting
Purpose. This study aimed to characterize A. baumannii strains isolated from patients in an intensive care unit (ICU) setting. Molecular techniques were used to study clonal relatedness and determine a fast, efficient and cost-effective way of detecting persistent clones.
Methodology. A. baumannii (n=17) were obtained in June and November 2015 from a single ICU setting in South India. DNA typing methods such as multilocus sequence typing (MLST), single-locus sequence-based typing (SBT) and DNA fingerprinting PCRs (M13, DAF4 and ERIC2) were employed to understand the association of clones. PCRs were performed for the antimicrobial resistance genes ISAba1-bla OXA-51-like, ISAba1-bla OXA-23-like, bla NDM-1, bla PER-7 and bla TEM-1, and the virulence genes cpa 1, cpa2 and pkf.
Results. The MLST showed some degree of corroboration with the other DNA typing methods. The M13 PCR was found to give better results than the other fingerprinting methods. ST848 (CC92) was the dominant strain isolated in both June and November. All isolates were bla OXA-51-like-positive, with 16 having ISAba1 upstream of the bla OXA-51-like and bla OXA-23-like genes. Genes such as bla NDM-1 (23 %, n=4), bla PER-7 (58.8 %, n=10), pkf (82 %, n=14), bla TEM-1 (5.8 %, n=1), cpa1 (5.8 %, n=1) and cpa2 (5.8 %, n=1) were also detected.
Conclusion. M13 PCR can be used in routine environmental surveillance for the detection of persistent antibiotic resistant clones in an ICU setting because of its reliability and simplicity. Further studies based on greater sample size, conducted at the multi-centre level, can give us a better understanding of the reliability of the molecular methods that can be used for the detection of persistent clones in the hospital setting.
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Mycoplasma amphoriforme vs M. pneumoniae: similarities and differences between patient characteristics in a regional hospital in the Netherlands
Mycoplasma amphoriforme is a species closely related to Mycoplasma pneumoniae, thus far with unknown clinical impact. The application of optimized diagnostics, better capable of differentiating between these two micro-organisms, identified a significant patient population positive for M. amphoriforme. The PCR designed by Ling et al. was used on respiratory samples that originally tested positive for M. pneumoniae (n=78), and identified 29 retrospectively as M. amphoriforme. The aim of this study is to describe and compare both groups. The group infected with M. amphoriforme was significantly older and more frequently had a co-infection (19 % vs 62 %), COPD and less fever. This could suggest that M. amphoriforme has opportunistic characteristics.
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- One Health
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A severe outbreak of listeriosis in central Italy with a rare pulsotype associated with processed pork products
Anna Duranti, Michela Sabbatucci, Giuliana Blasi, Vicdalia Aniela Acciari, Massimo Ancora, Antonino Bella, Luca Busani, Patrizia Centorame, Cesare Cammà, Fabrizio Conti, Dario De Medici, Marco Di Domenico, Violeta Di Marzio, Giovanni Filippini, Alfonsina Fiore, Stefano Fisichella, Antonietta Gattuso, Monica Gianfranceschi, Caterina Graziani, Fabrizia Guidi, Maurilia Marcacci, Cristina Marfoglia, Diana Neri, Massimiliano Orsini, Donatella Ottaviani, Annalisa Petruzzelli, Patrizio Pezzotti, Caterina Rizzo, Anna Ruolo, Gaia Scavia, Stefania Scuota, Giuliano Tagliavento, Alberto Tibaldi, Francesco Tonucci, Marina Torresi, Giacomo Migliorati and Francesco PomilioPurpose. From May 2015 to March 2016, an outbreak due to Listeria monocytogenes serotype 1/2a and clinical pulsotype never previously isolated in Europe occurred in central Italy, involving 24 confirmed clinical cases. The article provides a description of the outbreak and the investigation carried out by a multidisciplinary network.
Methodology. Epidemiological and microbiological surveillance was conducted to confirm the outbreak and to detect the food vehicle of infection. The origin and destination of the implicated food and its ingredients were investigated by tracing-back and -forward investigation.
Results. Next-generation sequencing confirmed the unique outbreak strain. On 4 January 2016, a L. monocytogenes strain with pulsotype indistinguishable from that isolated from clinical cases in the outbreak was detected in a sample of hog head cheese purchased from a retail supermarket by one of the patients. The hog head cheese was produced by a small meat processing plant in the Marche region, where microbiological investigation confirmed environmental and food contamination by the outbreak strain. Plant production was suspended and all contaminated batches of the hog head cheese were withdrawn from the market by 19 February by local health authority. We subsequently observed a sharp decline in clinical cases, the last being reported on 11 March 2016.
Conclusion. The key factor in the timely conclusion of this investigation was intersectoral collaboration among epidemiologists, microbiologists, veterinarians, statisticians and health and food safety authorities at national, regional and local levels.
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Characterization of the clonal subpopulation Fiocruz L1-130 of Leptospira interrogans in rats and dogs from Brazil
Purpose. Leptospira interrogans serogroup Icterohaemorrhagiae strains have been described as causing disease in both humans and animals and as being present worldwide. Icterohaemorrhagiae and Copenhageni serovars are known to cause severe disease in their hosts, and zoonotic outbreaks have been described. The genetic similarity among the strains of these serovars is known. However, it has not yet been demonstrated whether major clonal subpopulation in humans, strain Fiocruz L1-130-like, can circulate among other hosts.
Methodology. We performed genetic characterization of Brazilian serogroup Icterohaemorrhagiae strains of dog and rat origin by secY sequencing, variable-number tandem-repeat, multilocus sequence type and multi-spacer typing analysis.
Results. The strains were found to be identical among themselves and to strain Fiocruz L1-130. We suggest that the major strain of L. interrogans serogroup Icterohaemorrhagiae, Fiocruz L1-130, is widely distributed in Brazil in different hosts with substantial zoonotic potential.
Conclusion. Understanding the circulation of strain Fiocruz L1-130 is important for the implementation of appropriate control measures. Its circulation highlights the need to treat leptospirosis caused by L. interrogans serogroup Icterohaemorrhagiae as a zoonosis that acts in the human–animal–environment interface, as per the One Health approach.
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- Pathogenicity and Virulence/Host Response
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Comparative genome and evolution analysis of the locus of enterocyte effacement from enteropathogenic Escherichia coli Deng and its transcriptional response to ciprofloxacin
More LessPurpose. In this study, we aimed to investigate the genomic characteristics and evolution of pathogenicity islands of an enteropathogenic Escherichia coli (EPEC) strain, and to obtain a transcriptional profile of EPEC under different concentrations of ciprofloxacin using microarray analysis.
Methodology. The complete EPEC Deng genome was sequenced and compared to genomes of 12 previously sequenced E. coli strains. A 180 min time course experiment was performed in which the effect of ciprofloxacin on EPEC Deng growth was evaluated. Microarray profiling was used to study the effect of varying ciprofloxacin pressure on genome-wide transcriptional expression. Differential expression of the genes identified using microarray data was confirmed using real-time quantitative reverse transcriptase PCR (RTQ). Target gene-defective recombineering strains were created to investigate the influence of the grlA gene on ciprofloxacin susceptibility.
Results. Genomic comparisons revealed a close phylogenic relationship between EPEC Deng and E. coli strains O111_H_11128 and O26_H11_11368, with low genetic diversity among their type III secretion system genes and typically genetic variation in the map, tir, eae and espA genes of EPEC. It is noteworthy that 21 genes were down-regulated at all time points examined in the group exposed to 2 µg ml−1 of ciprofloxacin. A grlA-mutant derivative with increased susceptibility to ciprofloxacin was discovered.
Conclusions. The present findings provide an overview of the phylogenetic characteristics of EPEC Deng and its transcriptional response to ciprofloxacin, further suggesting that GrlA may play a clinically important role in EPEC responses to ciprofloxacin.
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- Prevention and Therapy
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Pasteurella multocida inactivated with ferric chloride and adjuvanted with bacterial DNA is a potent and efficacious vaccine in Balb/c mice
Purpose. Pasteurella multocida (P. multocida) is a principal pathogen of domestic animals and an opportunistic pathogen of humans. It is the causative agent of pneumonia and haemorrhagic septicaemia in cattle, sheep and goats, fowl cholera in chickens and progressive atrophic rhinitis in swine. In this study, we investigated the humoral and cellular immune responses and protective immunity conferred by an iron-inactivated vaccine with bacterial DNA (IIV+bDNA) as an adjuvant in mice.
Methodology. P. multocida was grown in BHI broth, inactivated with formalin and FeCl3 and adjuvanted with alum and bDNA. Mice were immunized with two whole-cell inactivated vaccine doses 2 weeks apart. The animals were challenged 4 weeks after booster immunization. Immunogens (vaccines and bDNA) posed no safety problems when mice were injected subcutaneously (s/c) with these preparations. The serum antibody titres were tested by ELISA. At 28 days post immunization, cell-mediated immunity responses were determined. The responses were measured by assay of IL-6 and IL-12 in lymphocyte spleen culture supernatants.
Results. ELISA results showed that the levels of antibodies in iron inactivated with bDNA adjuvant groups were higher than in the formalin inactivated with alum adjuvant vaccine group. The protection rate of IIV+bDNA adjuvant vaccine was superior to that of the other vaccines and it protected 100 % of the challenge group mice. Following immunization, bDNA promoted increased production of interleukins compared to the control groups.
Conclusion. These studies indicate that bDNA is effective as an immune adjuvant, and along with stimulatory bDNA represent promising new humoral and cellular immune enhancers for vaccination applications. In addition, this vaccine is able to provide long-term protection against infection.
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Fukugiside, a biflavonoid from Garcinia travancorica inhibits biofilm formation of Streptococcus pyogenes and its associated virulence factors
Purpose. Streptococcus pyogenes, a notorious human pathogen thatis responsible for various invasive and non-invasive diseases, possesses multiple virulence armaments, including biofilm formation. The current study demonstrates the anti-biofilm and anti-virulence potential of fukugiside, a biflavonoid isolated from Garciniatravancorica, against S. pyogenes.
Methodology. The anti-biofilm activity of fukugiside was assessed and established using microdilution and microscopic analysis. Biochemical assays were performed to assess the effects of fukugiside on important virulence factors, which were further validated using quantitative real-time PCR and in vivo analysis in Caenorhabditis elegans.
Results. Fukugiside exhibited concentration-dependent biofilm inhibition (79 to 96 %) against multiple M serotypes of S. pyogenes (M1, M56, M65, M74, M100 and st38) with a minimum biofilm inhibitory concentration of 80 µg ml−1. Electron microscopy and biochemical assay revealed a significant reduction in extracellular polymeric substance production. The results for the microbial adhesion to hydrocarbon assay, extracellular protease quantification and differential regulation of the dltA, speB, srv and ropB genes suggested that fukugiside probably inhibits biofilm formation by lowering cell surface hydrophobicity and destabilizing the biofilm matrix. The enhanced susceptibility to phagocytosis evidenced in the blood survival assay goes in unison with the downregulation of mga. The downregulation of important virulence factor-encoding genes such as hasA, slo and col370 suggested impaired virulence. In vivo analysis in C. elegans evinced the non-toxic nature of fukugiside and its anti-virulence potential against S. pyogenes.
Conclusion. Fukugiside exhibits potent anti-biofilm and anti-virulence activity against different M serotypes of S. pyogenes. It is also non-toxic, which augurs well for its clinical application.
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Time-kill kinetics of cadazolid and comparator antibacterial agents against different ribotypes of Clostridium difficile
More LessPurpose. Clostridium difficile infection (CDI) is an increasing cause of nosocomial diarrhoea worldwide, which has been partly attributed to the emergence of hypervirulent strains including C. difficile BI/NAP1/ribotype 027 and BK/NAP7/ribotype 078. Cadazolid is a new antibiotic currently in late-stage clinical studies for the treatment of CDI. The present study evaluated the in vitro bactericidal effect of cadazolid and comparator antibiotics against four C. difficile strains. The data demonstrate the potent and bactericidal activity of cadazolid against different ribotypes of C. difficile.
Methodology. MICs for test antibiotics were determined in brain– heart infusion-supplemented broth (BHIS) containing 5 g l−1 yeast extract and 0.025 % (w/v) l-cysteine. Time-kill kinetics to investigate the rate of killing of each antibiotic at sub- and supra-MIC concentrations were performed at concentrations of 0.5, 1, 2, 4, 8 or 16× the MIC of cadazolid, vancomycin and fidaxomicin at intervals over a 48 h period.
Results/key findings. Cadazolid-mediated killing of C. difficile was faster and occurred at lower concentrations than observed for vancomycin, while potency and killing was largely comparable to those observed for fidaxomicin. Notably, cadazolid also displayed a potent bactericidal effect against fluoroquinolone-resistant hypervirulent ribotype 027 and 078 strains. C. difficile spore formation was largely inhibited by all three antibiotics at concentrations >1× MIC; however, none were able to eliminate spores effectively, which were present at the start of the experiment.
Conclusion. The data presented here demonstrate the potent in vitro bactericidal activity of cadazolid against different ribotypes of C. difficile, although on a limited set of strains.
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The endogenous antiseptic N-chlorotaurine irreversibly inactivates Chlamydia pneumoniae and Chlamydia trachomatis
More LessPurpose. The antimicrobial activity of N-chlorotaurine (NCT), an endogenous long-lived oxidant applied topically, was tested against Chlamydiae in vitro.
Methodology. Elementary bodies of Chlamydia pneumoniae strain CV-6 and Chlamydia trachomatis serovars A and D were incubated in 0.01, 0.1 and 1 % (w/v) NCT solution at pH 7.1 and 37 °C. After different incubation times, aliquots were removed and grown in cell culture. The number of inclusion forming units was quantified by immunofluorescence and real-time qPCR.
Results/Key findings. Chlamydia pneumoniae and Chlamydia trachomatis were inactivated by 1 and 0.1 % NCT within 1 min. Moreover, 0.025–0.1 % NCT significantly reduced the number of intracellularly growing C. pneumoniae within 30 min.
Conclusions. This is the first study demonstrating the antimicrobial activity of NCT against Chlamydiae. Clinical implications of these findings have to be investigated in further trials.
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Volumes and issues
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 70 (2021)
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