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Volume 72,
Issue 6,
2023
Volume 72, Issue 6, 2023
- Editorials
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- Reviews
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Unravelling the flexibility of Mycobacterium tuberculosis: an escape way for the bacilli
The persistence of Mycobacterium tuberculosis makes it difficult to eradicate the associated infection from the host. The flexible nature of mycobacteria and their ability to adapt to adverse host conditions give rise to different drug-tolerant phenotypes. Granuloma formation restricts nutrient supply, limits oxygen availability and exposes bacteria to a low pH environment, resulting in non-replicating bacteria. These non-replicating mycobacteria, which need high doses and long exposure to anti-tubercular drugs, are the root cause of lengthy chemotherapy. Novel strategies, which are effective against non-replicating mycobacteria, need to be adopted to shorten tuberculosis treatment. This not only will reduce the treatment time but also will help prevent the emergence of multi-drug-resistant strains of mycobacteria.
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Insights into the challenging multi-country outbreak of Mpox: a comprehensive review
More LessHuman monkeypox virus (hMpoxV) is of zoonotic origin and is closely related to the once-dreaded smallpox virus. It is largely endemic to the African continent but has moved out of the endemic regions as sporadic clusters in the past 20 years, raising concerns worldwide. Human Mpox is characterized by a mild to severe, self-limiting infection, with mortality ranging from less than 1% to up to 10% during different outbreaks caused by different clades of MpoxV. Bushmeat hunting is one of the primary reasons for its transmission from animals to humans. Various international and national health regulatory bodies are closely monitoring the disease and have laid down guidelines to manage and prevent hMpox cases. Emergency Use Status has been granted to Tecovirimat and Brincidofovir to treat severe cases and vaccination with the smallpox vaccine is recommended for high-risk group individuals. Strategies to repurpose and discover novel therapeutics and vaccines to control the outbreak are being researched. The current Mpox outbreak that has mainly affected men as approximately 96% of all cases are reported in men, is probably the result of a complex intersection of various factors. This necessitates a strong One Health response coordination involving human, animal and environmental health institutions. This review is an attempt to provide an all-inclusive overview of the biology, history, epidemiology, pathophysiology, diagnosis and management of hMpox in context to the recent 2022–2023 multi-country outbreak which is termed by WHO a ‘Public Health Emergency of International Concern (PHEIC)’.
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- Antimicrobial Resistance
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Lipid A modification-induced colistin-resistant Klebsiella variicola from healthy adults
More LessBackground. Klebsiella variicola was once recognised as a benign plant-endosymbiont but recent case reports suggest that it is a newly emerging Gram-negative pathogen related to opportunistic infection of multiple sites in humans.
Methods. Antimicrobial susceptibility testing was performed using broth microdilution method. To identify colistin resistance mechanisms, phoPQ, pmrAB, and mgrB were sequenced and their mRNA expression was analysed using quantitative real-time PCR. In addition, we tried to detect crrAB and mcr. The lipid A moieties of colistin-susceptible and -resistant isolates were analysed using MALDI-TOF.
Results. Among the two K. variicola isolates, one is colistin-resistant, and another is colistin-susceptible. The colistin-resistant K. variicola isolate showed no mutations in phoPQ, pmrAB, and mgrB, and crrAB and mcr were not identified. However, its phoQ and pbgP expression was significantly higher and amino-arabinosylated lipid A with hexa-acylated species in lipopolysaccharide was identified.
Conclusions. We found that colistin resistance in K. variicola was mediated by the modification of lipid A. Although the isolate was obtained from faecal samples of healthy adults, colistin-resistant K. variicola challenges public health as an opportunistic pathogen.
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Detection of multiple extensively-drug resistant hypervirulent Klebsiella pneumoniae clones from patients with ventilator-associated pneumonia in Egypt
Introduction. Hypervirulent-K. pneumoniae (hvKP) is an evolving pathotype that is more virulent than the classical- K. pneumoniae (cKP) and causes serious fatal illnesses.
Hypothesis/Gap Statement. Although there are few reports on hvKP isolated from Egyptian patients, the molecular characteristics and clonal relatedness of MDR-hvKP have not been adequately investigated.
Aim. To investigate the microbiological and genetic characteristics as well as the epidemiology of hvKP induced ventilator-associated pneumonia (VAP).
Methodology. A retrospective study of 59 K . pneumoniae inducing VAP was conducted at Assiut University Hospitals from November 2017 to January 2019. All K. pneumoniae were tested for resistance phenotype, capsular genotype (K1 and K2), virulence gene profile (c-rmpA, p-rmpA, iucA, kfu, iroB, iroN), and the presence of resistance genes (blaNDM-1, blaCTX-M-3-like, blaCTX-M-14-like). Clonal relatedness was assessed by Pulsed field gel electrophoresis (PFGE).
Result. HvKP accounted for 89.8 % (53/59) of K. pneumoniae isolates with ~95 % exhibiting extensively-drug resistant (XDR) phenotype. Hypermucoviscous phenotype was detected in 19 (35.8 %) hvKP and K2 capsular gene was identified in 18 (33.9 %) of hvKP. Regarding the virulence genotype of hvKP strains, iucA was the most prevalent virulence gene (98.1%), while p-rmpA and kfu were detected in 75.4 and 52.8 % of hvKP strains, respectively. Resistance genes were highly prevalent in both cKP and hvKP with blaCTX-M-3-like being more prevalent in hvKP (100 % vs 94.3 % for blaNDM-1, 50 % vs 62.2 % for blaCTX-M-3- like and 83.3 % vs 69.8 % for blaCTX-M-14 -like, respectively). PFGE typing of 29 representative K. pneumoniae revealed 15 pulsotypes, with identical hvKP pulsotypes isolated from different ICUs at different times and several hvKP and cKP isolates belonged to the same pulsotype.
Conclusion. This study highlights the dominance and clonal spread of XDR-hvKP strains at Assiut University Hospital in Egypt. Physicians should be aware of the increased risk of hvKP induced-VAP and support further epidemiologic studies.
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Class 1 integrons in clinical and swine industry isolates of Salmonella Typhimurium from Colombia, dating 1997 to 2017
Background. Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) has been linked to outbreaks of foodborne gastroenteritis disease, and the emergence of antimicrobial-resistant clones. In Colombia, laboratory surveillance of Salmonella spp. between 1997–2018 revealed that S. Typhimurium was the most ubiquitous serovar (27.6 % of all Salmonella isolates), with increasing levels of resistance to several families of antibiotics.
Hypothesis. Resistant isolates of S. Typhimurium recovered from human clinical, food and swine samples carry class 1 integrons that are linked to antimicrobial resistance genes.
Aim. Identify class 1 integrons, and investigate their association with other mobile genetic elements, and their relationship to the antimicrobial resistance of Colombian S. Typhimurium isolates.
Methods. In this study, 442 isolates of S. Typhimurium were analysed, of which 237 were obtained from blood culture, 151 from other clinical sources, 4 from non-clinical sources and 50 from swine samples. Class 1 integrons and plasmid incompatibility groups were analysed by PCR and whole-genome sequencing (WGS), and regions flanking integrons were identified by WGS. The phylogenetic relationship was established by multilocus sequence typing (MLST) and single-nucleotide polymorphism (SNP) distances for 30 clinical isolates.
Results . Overall, 39 % (153/392) of the human clinical isolates and 22 % (11/50) of the swine S. Typhimurium isolates carried complete class 1 integrons. Twelve types of gene cassette arrays were identified, including dfr7-aac-bla OXA-2 (Int1-Col1), which was the most common one in human clinical isolates (75.2 %, 115/153). Human clinical and swine isolates that carried class 1 integrons were resistant to up to five and up to three antimicrobial families, respectively. The Int1-Col1 integron was most prevalent in stool isolates and was associated with Tn21. The most common plasmid incompatibility group was IncA/C.
Conclusions. The widespread presence of the IntI1-Col1 integron in Colombia since 1997 was striking. A possible relationship between integrons, source and mobile elements that favour the spread of antimicrobial resistance determinants in Colombian S. Typhimurium was identified.
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Temporal shifts in the predominant carbapenemase gene types among carbapenemase-producing Klebsiella pneumoniae isolated in Bangkok, Thailand, during 2013–2016
Introduction. Carbapenemase-producing Enterobacteriaceae (CPE) have emerged as a global threat to public health and clinical practice.
Hypothesis/Gap Statement. In Thailand, reports describing CPEs carrying bla NDM and bla OXA-48-like genes have been increasing recently; however, data on detailed plasmid analysis and temporal shift of sequence type and carbapenemase type are limited.
Aim. In this study, we analysed whole-genome sequencing (WGS) data of clinically isolated carbapenemase-producing Klebsiella pneumoniae (CPKP) to reveal the molecular epidemiology of CPKP in a tertiary-care hospital in Bangkok, Thailand.
Methodology. Seventy-seven non-duplicated CPKP isolates collected during 2013–2016 were examined for their drug-resistance genes, sequence types and phylogenetic relationships.
Results. All the tested isolates possessed carbapenemase gene(s), and the major type of carbapenemase gene in 2014–2015 was bla NDM-1, whereas isolates in 2016 harboured more bla OXA-232 than bla NDM-1. Other carbapenemase gene variants, such as bla NDM-4, bla NDM-5, bla OXA-48, bla OXA-181 and bla IMP-14 were detected in some CPKP isolates. Furthermore, this study revealed that CPKP co-harbouring two genes, bla NDM-1 and bla OXA-232 or bla OXA-181, emerged during this period. Notably, such isolates co-carrying the two carbapenemase genes emerged in three different sequence types, even in a single hospital, and then spread clonally. The WGS of CPKP revealed a temporal shift of the predominant carbapenemase genes from bla NDM-1 to bla OXA-232 along with a variation in other carbapenemase gene types within a span of 4 years.
Conclusion. Our findings suggest that a substantial change in CPE types occurred in Thailand and potentially in Southeast Asian countries.
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Antimicrobial resistance, molecular characteristics, virulence and pathogenicity of bla NDM-1-positive Enterobacter cloacae
Yan Yu, Pengfei Dai, Min Niu, Ruihui Han, Shumin Liu and Yan DuIntroduction. The bla NDM-1 -positive Enterobacter cloacae has led to limited therapeutic options for clinical treatment.
Hypothesis/Gap Statement. Analysing the antimicrobial resistance and molecular typing of bla NDM-1-positive E. cloacae is of great significance. Meanwhile, the effect of the bla NDM-1 gene on the virulence and pathogenicity of E. cloacae remains unclear and should be assessed.
Aim. To understand bla NDM-1-positive E. cloacae from different perspectives.
Methodology. The PCR was used to screen bla NDM-1-positive E. cloacae , then, antimicrobial susceptibility tests and multilocus sequence typing (MLST) were performed on them; sixty-nine strains of bla NDM-1-negative E. cloacae were collected as the controls, 28 pairs of virulence-related genes' carriage and biofilm-forming ability were detected for preliminary evaluation of the virulence phenotype of the strains; to gain insight into the effect of the bla NDM-1 gene on the virulence and pathogenicity of E. cloacae , the bla NDM-1-positive E. cloacae T2 (NDM-1), the T2 bla NDM-1 knockout strain (ΔNDM-1) and ATCC13047 (ST) were studied, compared the motility, anti-serum killing ability, and virulence to cells. Then, the mice intraperitoneal infection model was established, the survival curve, histopathological characteristics, bacterial load in spleen and the contents of cytokines were compared.
Results. (1) Thirty-five bla NDM-1-positive E. cloacae exhibited multidrug resistance. MLST distinguished 12 STs, ST74 was the most common clonal type (11/35), followed by ST114 (10/35). (2) The detection rates of virulence genes clpB, icmf, VasD/Lip and acrA in the bla NDM-1-positive E. cloacae were significantly higher than those in bla NDM-1-negative E. cloacae (P<0.05), while there was no significant difference in the amount of biofilm formation between two groups. (3) The presence of bla NDM-1 gene attenuated the motility diameter of E. cloacae , but had no significant effect on their ability to resist serum killing, and the virulence to cells. The survival rate, histopathological changes, bacterial load in spleen and inflammatory cytokines were not significantly affected.
Conclusions. (1) The bla NDM-1-positive E. cloacae exhibited multidrug resistance, and the MLST typing was mainly ST74 and ST114, with a small-scale clonal spread of the ST114 strain in the hospital NICU ward. (2) The bla NDM-1 gene did not affect the virulence and pathogenicity of E. cloacae .
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- Clinical Microbiology
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Epidemiology and urological pathogenic potential of Aerococcus species in greater Glasgow and Clyde (Descriptive study of Aerococcus urinae in blood culture and urinary samples: clinical importance and potential marker of urinary tract pathology)
More LessIntroduction. Aerococcus species in particular A. urinae are increasingly reported as causative agents of bacteraemia, urinary tract infection, sepsis, and endocarditis. We sought to establish the epidemiology of A. urinae in Glasgow hospitals and whether the presence of the organism in clinical isolates could be an indicator of undiagnosed urinary tract pathology.
Hypothesis/Gap statement. The knowledge gap among clinical staffs on Aerococcus species as emerging pathogens can be filled by understanding its epidemiology and clinical importance.
Aim. Describe the epidemiology and clinical importance of Aerococcus urinae .
Methodology. We reviewed positive blood cultures with Aerococcus species (2017–2021) and urinary isolates (2021) in Glasgow hospitals. Data were collected from clinical and laboratory database systems.
Results. All 22 positive blood cultures were A. urinae and sensitive to amoxicillin, vancomycin, and ciprofloxacin. The median age was 80.5; the majority was male (18). In total, 15/22 (68 %) were diagnosed with urinary tract infection. Thirteen were treated with amoxicillin. No cases of infective endocarditis were noted. One patient was subsequently diagnosed with bladder carcinoma. All 83 positive urinary isolates in 72 patients were A. urinae . One was resistant to amoxicillin; two to ciprofloxacin; all sensitive to nitrofurantoin and vancomycin. The majority was female (43/83), the median age was 80. The commonest risk factors were underlying malignancy including bladder cancer (5/18), chronic kidney disease (17) and diabetes (16). Clinical data was unavailable in 24 episodes. Of these, 41/59 (69.5 %) were diagnosed with urinary tract infection. One patient was subsequently diagnosed with metastatic renal cancer while bladder wall lesions were identified in three patients, two of whom were waiting for an urology review at the time of study. Thirteen patients (18 %) had 1 year recurrent bacteriuria and three were not treated on initial episode.
Conclusion. A. urinae are emerging pathogens and are likely to become more common due to advances in laboratory technologies and an ageing population. Clinical teams should be aware of their urological pathogenic potential and not dismiss them as contaminants. Whether Aerococcus infection is a potential indicator for undiagnosed urinary tract malignancy warrants further studies.
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Serotypes and antimicrobial resistance of Streptococcus pneumoniae in children under 5 years of age pre- and post-pneumococcal conjugate vaccine introduction in Paraguay
Introduction. Streptococcus pneumoniae remains a major cause of mortality and morbidity worldwide in children <5 years of age, even with advances in vaccination programmes.
Hypothesis/Gap Statement. Reviewing and reporting trends in the distribution of pneumococcal serotypes and antimicrobial resistance in Paraguay will be useful for decision-making in public health.
Aim. This study analysed the serotype distribution and antimicrobial resistance of S. pneumoniae and the characteristics of pneumococcal disease in children <5 years old before and after the introduction of pneumococcal conjugate vaccines (PCVs).
Methodology. A total of 885 isolates and 278 S. pneumoniae PCR-positive clinical specimens were referred to the Central Laboratory of Public Health (LCSP) within the meningitis and pneumonia laboratory based-surveillance network in the period 2006–2020. Conventional and molecular microbiological techniques were used for confirmation and characterization.
Results. We identified 563 cases of pneumococcal disease in the pre-vaccination period, 325 cases in the post-PCV10 period and 275 cases in the post-PCV13 period. The serotypes covered by PCV10 decreased from 78.6–6.5 %. However, additional serotypes covered by PCV13 increased from 6.6–57.5% and non-PCV13 serotypes increased from 14.8–36.0 % (P<0.001) in the post-PCV13 period. In cases of meningitis, the rate of resistance to penicillin decreased after the introduction of conjugate vaccines. No resistance to ceftriaxone was found in any period. In cases without meningitis, the rate of resistance to penicillin and ceftriaxone decreased slightly. However, the rate of resistance to erythromycin and tetracycline increased and that to trimethoprim–sulfamethoxazole (SXT) decreased in the post-PCV13 period compared to the pre-PCV period. The multidrug resistance rate was 8.5 %.
Conclusion. A change in the circulating serotypes and antimicrobial resistance to certain antibiotics was observed. Non-vaccine serotype circulation and multidrug resistance may compromise the success of the conjugate vaccines.
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A comparison of qPCR and microscopy for the detection and enumeration of Cryptosporidium oocysts from drinking water
More LessA corrigendum of this article has been published full details can be found at 10.1099/jmm.0.001926
Introduction. Cryptosporidium presents one of the main waterborne public health threats due to its resistance to chlorine disinfection and ability to cause large-scale outbreaks. The standard method used in the UK water industry for detection and enumeration of Cryptosporidium is based on fluorescence microscopy and is laborious and expensive. Molecular methods such as quantitative polymerase chain reaction (qPCR) can be more amenable to streamlining through automation, improving workflows and standardizing procedures.
Hypothesis. The null hypothesis was that there was no difference in the detection or enumeration between the standard method and a qPCR.
Aim. We aimed to develop and evaluate a qPCR for the detection and enumeration of Cryptosporidium in drinking water, and to compare the assay with the standard method used in the UK.
Methodology. We first developed and evaluated a qPCR method by incorporating an internal amplification control and calibration curve into a real-time PCR currently used for Cryptosporidium genotyping. Then we compared the qPCR assay with the standard method of immunofluorescent microscopy for the detection and enumeration of 10 and 100 Cryptosporidium oocysts in 10 l of artificially contaminated drinking water.
Results. The results demonstrated that detection of Cryptosporidium by this qPCR was reliable at low numbers of oocysts; however, enumeration was less reliable and more variable than immunofluorescence microscopy.
Conclusions. Despite these results, qPCR offers practical advantages over microscopy. There is potential for the use of PCR-based methods for Cryptosporidium analysis if parts of the upstream sample preparation are revised, and alternative technologies for enumeration (such as digital PCR) are also explored to improve analytical sensitivity.
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Household transmission of non-toxigenic diphtheria toxin gene-bearing Corynebacterium diphtheriae following a cluster of cutaneous cases in a specialist outpatient setting
Norman K. Fry, Ellen Pringle, William Newsholme, Margot Nicholls, Jim Stephenson, Rachel Thorn Heathcock, Charlotte Gower, Joanne Lacy, Shennae O’Boyle, David J. Litt, Carmen Sheppard, Natalie Groves, Joshua D’Aeth, Katie L. Hopkins, Danièle Meunier, Aruni De Zoysa, Androulla Efstratiou, Colin Brown, Meera Chand and Gayatri AmirthalingamIntroduction. Combination of PCR and Elek testing to identify toxigenic corynebacteria has revealed organisms described as non-toxigenic toxin-gene bearing (NTTB) Corynebacterium diphtheriae or C. ulcerans (i.e. PCR tox positive; Elek negative). These organisms carry part or all of tox, but are unable to express diphtheria toxin (DT) and present a challenge to clinical and public health case management.
Gap analysis/Hypothesis. There are few data on the theoretical risk of NTTB reversion to toxigenicity. This unique cluster and subsequent epidemiologically linked isolates allowed the opportunity to determine any change in DT expression status.
Aim. To characterize a cluster of infections due to NTTB in a skin clinic and subsequent cases in two household contacts.
Methodology. Epidemiological and microbiological investigations were carried out according to existing national guidance at the time. Susceptibility testing used gradient strips. The tox operon analysis and multi-locus sequence typing (MLST) was derived from whole-genome sequencing. Alignment of the tox operon and phylogenetic analyses were performed using clustalW, mega, the public core-genome MLST (cgMLST) scheme and an in-house bioinformatic single nucleotide polymorphism (SNP) typing pipeline.
Results. Isolates of NTTB C. diphtheriae were recovered from four cases (cases 1 to 4) with epidermolysis bullosa attending the clinic. Two further isolates were subsequently recovered from case 4, >18 months later, and from two household contacts (cases 5 and 6) after a further 18 months and 3.5 years, respectively. All eight strains were NTTB C. diphtheriae biovar mitis, belonged to the same sequence type (ST-336) with the same deletion in tox. Phylogenetic analysis showed relatively high diversity between the eight strains with 7–199 SNP and 3–109 cgMLST loci differences between them. The number of SNPs between the three isolates from case 4 and two household contacts (cases 5 and 6) was 44–70 with 28–38 cgMLST loci differences.
Conclusions. We report a cluster of NTTB C. diphtheriae cases in a skin clinic and evidence of onward household transmission. We conclude the deletion in the tox was responsible for the non-expression of DT. There was no evidence of reversion to DT expression over the 6.5 year period studied. These data informed revision to guidance in the management of NTTB cases and their contacts in the UK.
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- Disease, Diagnosis and Diagnostics
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Escherichia coli encoding Shiga toxin subtype Stx2f causing human infections in England, 2015–2022
Introduction. Shiga toxin-producing Escherichia coli (STEC) belong to a diverse group of gastrointestinal pathogens defined by the presence of Shiga toxin genes (stx) of which there are at least ten subtypes (Stx1a-Stx1d and Stx2a-Stx2g).
Gap Statement. Initially thought to be associated with mild symptoms, more recently STEC encoding stx2f have been isolated from cases of haemolytic uraemic syndrome (HUS) and the clinical significance and public health burden require further investigation.
Aim. We analysed clinical outcomes and genome-sequencing data linked to patients infected with STEC encoding-stx2f in England to assess the risk to public health.
Methodology. One hundred and twelve E. coli (n=58 isolates encoded stx2f; n=54 isolates E. coli belonging to CC122 or CC722 that had eae but were negative for stx) isolated from patients' faecal specimens between 2015 and 2022 were genome sequenced and linked to epidemiological and clinical outcome data. All isolates were investigated for the presence of virulence genes and a maximum-likelihood phylogeny of isolates belonging to CC122 and CC722 was constructed.
Results. There were 52 cases infected with STEC harbouring stx2f between 2015 and 2022, with the majority identified in 2022. Most cases resided in the North of England (n=39/52, 75 %), were female (n=31, 59.6 %) and/or aged five and under (n=29, 55.8 %). Clinical outcome data were available for 40/52 cases (76.9 %) and 7/40(17.5 %) were diagnosed with STEC-HUS. In the two most common clonal complexes, CC122 and CC722, the presence of the stx2f-encoding prophage correlated with the presence of additional virulence genes, astA, bfpA and cdt, located on an 85kbp IncFIB plasmid.
Conclusions. Certain serotypes of E. coli harbouring stx2f cause severe clinical outcomes, including STEC-HUS. Public health advice and possible interventions are limited, as little is known about the animal and environmental reservoirs and transmission routes. We recommend more comprehensive and standardized collection of microbiological and epidemiological data, and routine sharing of sequencing data between public health agencies worldwide.
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The bacteriology of diabetic foot ulcers and infections and incidence of Staphylococcus aureus Small Colony Variants
Introduction. Uninfected diabetes-related foot ulcer (DFU) progression to diabetes-related foot infection (DFI) is a prevalent complication for patients with diabetes. DFI often progresses to osteomyelitis (DFI-OM). Active (growing) Staphylococcus aureus is the most common pathogen in these infections. There is relapse in 40–60 % of cases even when the initial treatment at the DFI stage apparently clears infection.
Hypothesis. S. aureus adopts the quasi-dormant Small Colony Variant (SCV) state during DFU and consequently infection, and when present in DFI cases also permits survival in non-diseased tissues as a reservoir to cause relapse.
Aim. The aim of this study was to investigate the bacterial factors that facilitate persistent infections.
Methodology. People with diabetes were recruited from two tertiary hospitals. Clinical and bacterial data was taken from 153 patients with diabetes (51 from a control group with no ulcer or infection) and samples taken from 102 patients with foot complications to identify bacterial species and their variant colony types, and then compare the bacterial composition in those with uninfected DFU, DFI and those with DFI-OM, of whom samples were taken both from wounds (DFI-OM/W) and bone (DFI-OM/B). Intracellular, extracellular and proximal ‘healthy’ bone were examined.
Results. S. aureus was identified as the most prevalent pathogen in diabetes-related foot pathologies (25 % of all samples). For patients where disease progressed from DFU to DFI-OM, S. aureus was isolated as a diversity of colony types, with increasing numbers of SCVs present. Intracellular (bone) SCVs were found, and even within uninfected bone SCVs were present. Wounds of 24 % of patients with uninfected DFU contained active S. aureus . All patients with a DFI with a wound but not bone infection had previously had S. aureus isolated from an infection (including amputation), representing a relapse.
Conclusion. The presence of S. aureus SCVs in recalcitrant pathologies highlights their importance in persistent infections through the colonization of reservoirs, such as bone. The survival of these cells in intracellular bone is an important clinical finding supporting in vitro data. Also, there seems to be a link between the genetics of S. aureus found in deeper infections compared to those only found in DFU.
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Immunoassay-based evaluation of rOmp28 protein as a candidate for the identification of Brucella species
More LessIntroduction. Brucellosis is an important bacterial zoonosis, re-emerging as a serious public health concern in developing countries. Two major species, Brucella melitensis and Brucella abortus , cause recurrent facile infection in human. Therefore, rapid and accurate diagnosis for early disease control and prevention is needed in areas with low disease burden.
Hypothesis. This study evaluated the sandwich enzyme-linked immunosorbent assay (ELISA) (S-ELISA) immunoassay for potential use of whole-cell (WC) and recombinant outer-membrane protein (rOmp28)-derived IgG polyclonals in sensitive detection of Brucella .
Aim. Immunoassay-based WC detection of Brucella species in important sub-clinical matrices at lower limits of detection.
Methodology. We purified recombinant rOmp28 with Ni–NTA gel affinity chromatography and produced IgG polyclonal antibodies (pAbs) using BALB/c mice and New Zealand white female rabbits against different antigens (Ags) of Brucella . Checkerboard sandwich ELISA and P/N ratio (optical density of ‘P’ positive test sample to ‘N’ negative control) were used for evaluation and optimization of the study. The pAbs were characterized using Western blot analysis and different matrices were spiked with WC Ag of Brucella .
Results. Double-antibody S-ELISA was developed using WC Ag-derived rabbit IgG (capture antibody at 10 µg ml−1) and rOmp28-derived mice IgG (detection antibody at 100 µg ml−1) with a detection range of 102 to 108 cells ml−1 and a limit of detection at 102 cells ml−1. A P/N ratio of 1.1 was obtained with WC pAbs as compared to 0.6 and 0.9 ratios with rOmp28-derived pAbs for detecting B. melitensis 16M and B. abortus S99, respectively. An increased P/N ratio of 4.4 was obtained with WC Ag-derived rabbit IgG as compared to 4.2>4.1>2.4 ratios obtained with rabbit IgGs derived against cell envelope (CE), rOmp28 and sonicated antigen (SA) of Brucella with high affinity for rOmp28 Ag analysed on immunoblots. The rOmp28-derived mice IgG revealed two Brucella species at P/N ratios of 11.8 and 6.3, respectively. Upon validation, S-ELISA detected Brucella WCs in human whole blood and sera samples with no cross-reactivity to other related bacteria.
Conclusion. The developed S-ELISA is specific and sensitive in early detection of Brucella from different matrices of clinical and non-clinical disease presentation.
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Development and evaluation of Microbe Finder (MiFi)®: a novel in silico diagnostic platform for pathogen detection from metagenomic data
More LessIntroduction. With expanding demand for diagnostics, newer methodologies are needed for faster, user-friendly and multiplexed pathogen detection. Metagenome-based diagnostics offer potential solutions to address these needs as sequencing technologies have become affordable. However, the diagnostic utility of sequencing technologies is currently limited since analysis of the large amounts of data generated, are either computationally expensive or carry lower sensitivity and specificity for pathogen detection.
Hypothesis/Gap Statement. There is a need for novel, user friendly, and computationally inexpensive platforms for metagenome sequence analysis for diagnostic applications.
Methods. In this study, we report the use of MiFi® (Microbe Finder), a computationally inexpensive algorithm with a user-friendly online interface, for accurate, rapid and multiplexed pathogen detection from metagenome sequence data. Detection is accomplished based on identification of signature genomic sequence segments of the target pathogen in metagenome sequence data. In this study we used bovine respiratory disease (BRD) complex as a model.
Results and Conclusions. Using MiFi®, multiple target bacteria and a DNA virus were successfully detected in a multiplex format from metagenome sequences acquired from bovine lung tissue. Overall, 51 clinical samples were assessed and MiFi® showed 100 % analytical specificity and varying levels of analytical sensitivity (62.5 %–100 %) when compared with other traditional pathogen detection techniques, such as PCR. Consistent detection of bacteria was possible from lung samples artificially spiked with 109–104 c.f.u. of Mannheimia haemolytica .
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Evaluation and validation of a commercial ELISA versus the in vitro toxin neutralization assay for determination of diphtheria anti-toxin in human serum
Introduction. Diphtheria is a potentially life-threatening infection and remains endemic in many low- and middle-income countries (LMICs). A reliable, low-cost method for serosurveys in LMICs is warranted to estimate the accurate population immunity to control diphtheria.
Hypothesis/Gap Statement. The correlation between the ELISA results against diphtheria toxoid and the gold standard diphtheria toxin neutralization test (TNT) values is poor when ELISA values are <0.1 IU ml−1, which results in inaccurate estimates of susceptibility in populations when ELISA is used for measuring antibody levels.
Aim. To explore methods to accurately predict population immunity and TNT-derived anti-toxin titres from ELISA anti-toxoid results.
Methodology. A total of 96 paired serum and dried blood spot (DBS) samples collected in Vietnam were used for comparison of TNT and ELISA. The diagnostic accuracy of ELISA measurement with reference to TNT was assessed by area under the receiver operating characteristic (ROC) curve (AUC) and other parameters. Optimal ELISA cut-off values corresponding to TNT cut-off values of 0.01 and 0.1 IU ml−1 were identified by ROC analysis. A method based on the multiple imputation approach was also applied to estimate TNT measurements in a dataset that only included ELISA results. These two approaches were then applied to ELISA results previously generated from 510 subjects in a serosurvey in Vietnam.
Results. The ELISA results on DBS samples showed a good diagnostic performance compared to TNT. The cut-off values for ELISA measurement corresponding to the TNT cut-off values of 0.01 IU ml−1 were 0.060 IU ml−1 in serum samples, and 0.044 IU ml−1 in DBS samples. When a cut-off value of 0.06 IU ml−1 was applied to the 510 subject serosurvey data, 54 % of the population were considered susceptible (<0.01 IU ml−1). The multiple imputation-based approach estimated that 35 % of the population were susceptible. These proportions were much larger than the susceptible proportion estimated by the original ELISA measurements.
Conclusion. Testing a subset of sera by TNT combined with ROC analysis or a multiple imputation approach helps to adjust ELISA thresholds or values to assess population susceptibility more accurately. DBS is an effective low-cost alternative to serum for future serological studies for diphtheria.
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Changes in serum amyloid A, plasma high-density lipoprotein cholesterol and apolipoprotein A-I as useful biomarkers for Mycobacterium tuberculosis infection
Introduction. In recent years, cholesterol has received interest in the study of infection due to evidence of a relationship between low plasma cholesterol levels and tuberculosis (TB).
Hypothesis/Gap Statement. Plasma lipid profiles of serum amyloid A (SAA), apolipoprotein A-I and high-density lipoprotein cholesterol (HDL-C) are biomarkers associated with symptomatic TB patients.
Objective. We aimed to evaluate plasma lipid profiles of apolipoprotein A-I, SAA and the size of HDL as biomarkers to diagnose symptomatic TB patients.
Methodology. Patients with TB symptoms attending the Instituto Brasileiro para a Investigação da Tuberculose/Fundação José Silveira (IBIT/FJS) between September 2015 and August 2016 for diagnosis of TB were studied. From 129 patients, 97 were classified as pulmonary TB and 32 as negative-bacilloscopy (non-TB group). Medical history, fasting serum and plasma were obtained. Total cholesterol (TC), HDL-C, apolipoprotein A-I and SAA were measured by enzymatic or immunochemical reaction assays. HDL size was measured by laser light-scattering.
Results. In TB patients, TC (147.0±37 vs. 168±44 mg dL−1), HDL-C (37±14 vs. 55±18 mg dL−1) and apolipoprotein A-I (102±41 vs. 156±47 mg dL−1) concentrations were lower (P<0.0001), while HDL particle size (10.16±1.02 vs. 9.62±0.67 nm) and SAA levels (280±36 vs. 19±8 mg L−1) were higher (P<0.0001). Using receiver-operating characteristic curve analysis for predicting TB, the cutoff values were <83.85 mg L−1 for SAA (sensitivity=96.88 %, specificity=78.43 %, P<0.0001), >44.50 mg dL−1 for HDL-C (sensitivity=75 %, specificity=72.16 %, P<0.001) and >118.5 mg dL−1 for apolipoprotein A-I (sensitivity=83.83 %, specificity=72.22 %, P<0.001).
Conclusion. SAA, HDL-C and apolipoprotein A-I are associated with TB infection and could be used as laboratory biomarkers, especially in patients who are negative for alcohol-acid-resistant bacilli.
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- Medical Mycology
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CLEC4A and CLEC12B C-type lectin receptors mediate interactions with Pneumocystis cell wall components
More LessIntroduction. C-type lectin receptors (CLRs) are prominently expressed on myeloid cells where they perform multiple functions including serving as pattern recognition receptors (PRRs) to drive innate as well as adaptive immunity to pathogens. Depending on the presence of a tyrosine-based signalling motif, CLR-microbial pathogen engagement may result in either anti- or pro-inflammatory signalling.
Impact statement. In this manuscript, we report our laboratory study of two novel CLRs that recognize Pneumocystis murina cell wall homogenates (CWH) and a purified Pneumocystis carinii cell wall fraction (CWF).
Aim. To study the potential of newly generated hFc-CLR fusions on binding to Pneumocystis murina CWHs and P. carinii CWFs and subsequent downstream inflammatory signalling analysis.
Methods. Newly generated hFc-CLR fusion CLEC4A and CLEC12B were screened against P. murina CWHs and P. carinii CWFs preparations via modified ELISA. Immunofluorescence assay (IFA) was utilized to visualize hFc-CLR fusion binding against intact fixed fungal life forms to verify results. Quantitative PCR (q-PCR) analysis of lung mRNA from the mouse immunosuppressed Pneumocystis pneumonia (PCP) model versus uninfected mice was employed to detect possible changes in the respective Clec4a and Clec12b transcripts. Lastly, siRNA technology of both CLRs was conducted to determine effects on downstream inflammatory events in mouse macrophages stimulated in the presence of P. carinii CWFs.
Results. We determined that both CLEC4A and CLEC12B hFc-CLRs displayed significant binding with P. murina CWHs and P. carinii CWFs. Binding events showed significant binding to both curdlan and laminarin, both polysaccharides containing β-(1,3) glucans as well as N-acetylglucosamine (GlcNAc) residues and modest yet non-significant binding to the negative control carbohydrate dextran. IFA with both CLR hFc-fusions against whole P. murina life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of both CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that both CLRs were significantly up regulated during infection. Lastly, siRNA of both CLRs in the mouse RAW macrophage cell line was conducted and results demonstrated that silencing of Clec4a resulted in no significant changes in TNF-alpha generation in P. carinii CWF stimulated macrophages. On the contrary, silencing of Clec12b CLR resulted in significant decreases in TNF-alpha in RAW cells stimulated with the same CWF.
Conclusion. The data presented here provide new members of the CLRs family recognizing Pneumocystis. Future studies using CLEC4A and/or CLEC12B deficient mice in the PCP mouse model should provide further insights into the host immunological response to Pneumocystis.
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- Microbiome and Microbial Ecology in Health
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Using gut microbiota as a diagnostic tool for colorectal cancer: machine learning techniques reveal promising results
More LessIntroduction. Increasing evidence suggests a correlation between gut microbiota and colorectal cancer (CRC).
Hypothesis/Gap Statement. However, few studies have used gut microbiota as a diagnostic biomarker for CRC.
Aim. The objective of this study was to explore whether a machine learning (ML) model based on gut microbiota could be used to diagnose CRC and identify key biomarkers in the model.
Methodology. We sequenced the 16S rRNA gene from faecal samples of 38 participants, including 17 healthy subjects and 21 CRC patients. Eight supervised ML algorithms were used to diagnose CRC based on faecal microbiota operational taxonomic units (OTUs), and the models were evaluated in terms of identification, calibration and clinical practicality for optimal modelling parameters. Finally, the key gut microbiota was identified using the random forest (RF) algorithm.
Results. We found that CRC was associated with the dysregulation of gut microbiota. Through a comprehensive evaluation of supervised ML algorithms, we found that different algorithms had significantly different prediction performance using faecal microbiomes. Different data screening methods played an important role in optimization of the prediction models. We found that naïve Bayes algorithms [NB, accuracy=0.917, area under the curve (AUC)=0.926], RF (accuracy=0.750, AUC=0.926) and logistic regression (LR, accuracy=0.750, AUC=0.889) had high predictive potential for CRC. Furthermore, important features in the model, namely s__metagenome_g__Lachnospiraceae_ND3007_group (AUC=0.814), s__Escherichia_coli_g__Escherichia-Shigella (AUC=0.784) and s__unclassified_g__Prevotella (AUC=0.750), could each be used as diagnostic biomarkers of CRC.
Conclusions. Our results suggested an association between gut microbiota dysregulation and CRC, and demonstrated the feasibility of the gut microbiota to diagnose cancer. The bacteria s__metagenome_g__Lachnospiraceae_ND3007_group, s__Escherichia_coli_g__Escherichia-Shigella and s__unclassified_g__Prevotella were key biomarkers for CRC.
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