- Volume 70, Issue 3, 2021
Volume 70, Issue 3, 2021
- Editorial
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2020: the year of living cautiously
More Less2020 was the year when microbiology burst onto the world stage, not just as the science of small living things, but as the prism through which we understood global events. Clinical logic suffered under pressure arising from an urgent need to confirm or exclude severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This is a generation’s Hobbesian moment in which the public concern for safety and security from infection outweighs the pursuit of personal freedom. The strangeness of a world in which a minute particle wields superhuman power has generated its list of unlikely heroes and mendacious villains. As the year comes to an end, there are glimmers of light amid the gloom: the prospect of an effective vaccine, and life after the pandemic.
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- Review
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Genomic investigation of clinically significant coagulase-negative staphylococci
More LessIntroduction. Coagulase-negative staphylococci have been recognized both as emerging pathogens and contaminants of clinical samples. High-resolution genomic investigation may provide insights into their clinical significance.
Aims. To review the literature regarding coagulase-negative staphylococcal infection and the utility of genomic methods to aid diagnosis and management, and to identify promising areas for future research.
Methodology. We searched Google Scholar with the terms ( Staphylococcus ) AND (sequencing OR (infection)). We prioritized papers that addressed coagulase-negative staphylococci, genomic analysis, or infection.
Results. A number of studies have investigated specimen-related, phenotypic and genetic factors associated with colonization, infection and virulence, but diagnosis remains problematic.
Conclusion. Genomic investigation provides insights into the genetic diversity and natural history of colonization and infection. Such information allows the development of new methodologies to identify and compare relatedness and predict antimicrobial resistance. Future clinical studies that employ suitable sampling frames coupled with the application of high-resolution whole-genome sequencing may aid the development of more discriminatory diagnostic approaches to coagulase-staphylococcal infection.
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Sampling challenges in diagnosis of chronic bacterial infections
In recent decades there has been an increase in knowledge of the distribution, species diversity and growth patterns of bacteria in human chronic infections. This has challenged standard diagnostic methods, which have undergone a development to both increase the accuracy of testing as well as to decrease the occurrence of contamination. In particular, the introduction of new technologies based on molecular techniques into the clinical diagnostic process has increased detection and identification of infectious pathogens. Sampling is the first step in the diagnostic process, making it crucial for obtaining a successful outcome. However, sampling methods have not developed at the same speed as molecular identification. The heterogeneous distribution and potentially small number of pathogenic bacterial cells in chronic infected tissue makes sampling a complicated task, and samples must be collected judiciously and handled with care. Clinical sampling is a step in the diagnostic process that may benefit from innovative methods based on current knowledge of bacteria present in chronic infections. In the present review, we describe and discuss different aspects that complicate sampling of chronic infections. The purpose is to survey representative scientific work investigating the presence and distribution of bacteria in chronic infections in relation to various clinical sampling methods.
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On the emergence, spread and resistance of Candida auris: host, pathogen and environmental tipping points
More LessOver a decade ago, a multidrug-resistant nosocomial fungus Candida auris emerged worldwide and has since become a significant challenge for clinicians and microbiologists across the globe. A resilient pathogen, C. auris survives harsh disinfectants, desiccation and high-saline environments. It readily colonizes the inanimate environment, susceptible patients and causes invasive infections that exact a high toll. Prone to misidentification by conventional microbiology techniques, C. auris rapidly acquires multiple genetic determinants that confer multidrug resistance. Whole-genome sequencing has identified four distinct clades of C. auris, and possibly a fifth one, in circulation. Even as our understanding of this formidable pathogen grows, the nearly simultaneous emergence of its distinct clades in different parts of the world, followed by their rapid global spread, remains largely unexplained. We contend that certain host–pathogen–environmental factors have been evolving along adverse trajectories for the last few decades, especially in regions where C. auris originally appeared, until these factors possibly reached a tipping point to compel the evolution, emergence and spread of C. auris. Comparative genomics has helped identify several resistance mechanisms in C. auris that are analogous to those seen in other Candida species, but they fail to fully explain how high-level resistance rapidly develops in this yeast. A better understanding of these unresolved aspects is essential not only for the effective management of C. auris patients, hospital outbreaks and its global spread but also for forecasting and tackling novel resistant pathogens that might emerge in the future. In this review, we discuss the emergence, spread and resistance of C. auris, and propose future investigations to tackle this resilient pathogen.
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The prevalence of dermatophytoses in Brazil: a systematic review
Dermatophytosis is a common cutaneous mycosis worldwide whose prevalence in Brazil is still unknown. This systematic review has estimated the burden of dermatophytoses from updated literature data reported in the general Brazilian population. We used the following databases: Web of Science, Medline/PubMed, Embase, The Cochrane Library and Scopus for studies published between 2011 and 2020. Original articles with an emphasis on prevalence data for dermatophytosis in the Brazilian population, and diagnosed by culture exam or molecular biology were eligible. We also assessed the methodological quality of the studies. A total of 24 articles met the inclusion criteria and were reviewed. The occurrence of dermatophytoses found in the studies ranged from 4–88.50 %. The pooled prevalence of dermatophytosis for the population studies was 25 % (95 % CI: 24.7–25.3 %). The size of the samples used in the studies ranged from 45 to 36 446 participants, and ages ranged up to 98 years old. The populations studied involved mostly women. The presence of tinea unguium (toenail and fingernail) and tinea pedis were the most frequent dermatophytosis, and we observed a predominance of Trichophyton rubrum, T. interdigitale and T. mentagrophytes. The studies were primarily conducted in patient groups with suspected mycoses and were not entirely representative of the general population. Yet we believe that in the future, more collaborative strategies would improve both diagnostic capacity and epidemiological methodologies, associating the prevalence of dermatophytosis with social and environmental risk factors. This review helps to better understand future epidemiological trends in Brazil and the world.
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Pathogenicity and drug resistance of animal streptococci responsible for human infections
More LessBacteria of the genus Streptococcus , earlier considered typically animal, currently have also been causing infections in humans. It is necessary to make clinicians aware of the emergence of new species that may cause the development of human diseases. There is an increasing frequency of isolation of streptococci such as S. suis , S. dysgalactiae , S. iniae and S. equi from people. Isolation of Streptococcus bovis/Streptococcus equinus complex bacteria has also been reported. The streptococcal species described in this review are gaining new properties and virulence factors by which they can thrive in new environments. It shows the potential of these bacteria to changes in the genome and the settlement of new hosts. Information is presented on clinical cases that concern streptococcus species belonging to the groups Bovis, Pyogenic and Suis. We also present the antibiotic resistance profiles of these bacteria. The emerging resistance to β-lactams has been reported. In this review, the classification, clinical characteristics and antibiotic resistance of groups and species of streptococci considered as animal pathogens are summarized.
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- JMM Profile
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JMM Profile: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
More LessThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the cause of an infection known as coronavirus infectious disease 2019 (COVID-19). COVID-19 has become a global source of morbidity, mortality and social disruption since its emergence in East Asia in late 2019 and subsequent pandemic spread. Typical symptoms include cough, sore throat, fever, and sudden loss of taste and smell. Persistent, post-infection sequelae have been noted in a minority of cases. Severe complications and deaths occur mostly in older adults. Laboratory confirmation can be performed by viral RNA and antigen detection in nasal swabs or by detecting specific neutralizing antibodies. There is no effective and approved antiviral treatment, but several vaccines with favourable safety and efficacy profiles are being used in mass vaccination programmes. Vaccine-based COVID control should be seen as an addition to existing hygiene measures such as physical distancing, increased hand hygiene, cough etiquette, and barrier protection with personal protective equipment for frontline healthcare workers and other high-risk professions.
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- Antimicrobial Resistance
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Novel multiplex PCRs for detection of the most prevalent carbapenemase genes in Gram-negative bacteria within Germany
Introduction. Gram-negative bacteria are a common source of infection both in hospitals and in the community, and antimicrobial resistance is frequent among them, making antibiotic therapy difficult, especially when these isolates carry carbapenem resistance determinants.
Hypothesis/Gap Statement. A simple method to detect all the commonly found carbapenemases in Germany was not available.
Aim. The aim of this study was to develop a multiplex PCR for the rapid and reliable identification of the most prevalent carbapenemase-encoding genes in Gram-negative bacteria in Germany.
Methodology. Data from the German Gram-negative reference laboratory revealed the most prevalent carbapenemase groups in Germany were (in order of prevalence): bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM, bla OXA-40, bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51, bla IMI, bla FIM and bla DIM. We developed and tested two multiplex PCRs against 83 carbapenem-resistant Gram-negative clinical isolates. Primers were designed for each carbapenemase group within conserved regions of the encoding genes obtained from publicly available databases. Multiplex-1 included the carbapenemase groups bla VIM, bla OXA-48, bla OXA-23, bla KPC, bla NDM and bla OXA-40, while multiplex-2 included bla OXA-58, bla IMP, bla GIM, bla GES, ISAba1-bla OXA-51 and bla IMI.
Results. In the initial evaluation, all but one of the carbapenemases encoded by 75 carbapenemase-positive isolates were detected using the two multiplex PCRs, while no false-positive results were obtained from the remaining eight isolates. After evaluation, we tested 546 carbapenem-resistant isolates using the multiplex PCRs, and all carbapenemases were detected.
Conclusion. A rapid and reliable method was developed for detection and differentiation of 12 of the most prevalent carbapenemase groups found in Germany. This method allows for the rapid testing of clinical isolates prior to species identification and does not require prior phenotypical characterization, constituting a rapid and valuable tool in the management of infections in hospitals.
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Emergence of mgrB locus deletion mediating polymyxin resistance in pandemic KPC-producing Klebsiella pneumoniae ST15 lineage
Klebsiella pneumoniae causes a diversity of infections in both healthcare and community settings. This pathogen is showing an increased ability to accumulate antimicrobial resistance and virulence genes, making it a public health concern. Here we describe the whole-genome sequence characteristics of an ST15 colistin-resistant K. pneumoniae isolate obtained from a blood culture of a 79-year-old female patient admitted to a university hospital in Brazil. Kp14U04 was resistant to most clinically useful antimicrobial agents, remaining susceptible only to aminoglycosides and fosfomycin. The colistin resistance in this isolate was due to a ~1.3 kb deletion containing four genes, namely mgrB, yebO, yobH and the transcriptional regulator kdgR. The study isolate presented a variety of antimicrobial resistance genes, including the carbapenemase-encoding gene bla KPC-2, the extended-spectrum beta-lactamase (ESBL)-encoding gene bla SHV-28 and the beta-lactamase-encoding gene bla OXA-1. Additionally, Kp14U04 harboured a multiple stress resistance protein, efflux systems and regulators, heavy metal resistance and virulence genes, plasmids, prophage-related sequences and genomic islands. These features revealed the high potential of this isolate to resist antimicrobial therapy, survive in adverse environments, cause infections and overcome host defence mechanisms.
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Emergence of carbapenem-resistant Klebsiella pneumoniae harbouring bla OXA-48-like genes in China
Klebsiella pneumoniae strains carrying OXA-48-like carbapenemases are increasingly prevalent across the globe. There is thus an urgent need to better understand the mechanisms that underpin the dissemination of bla OXA-48-like carbapenemases. To this end, four ertapenem-resistant K. pneumoniae isolates producing OXA-48-like carbapenemases were isolated from two patients. Genome sequencing revealed that one sequence type (ST) 17 isolate carried bla OXA-181, whilst three isolates from a single patient, two ST76 and one ST15, carried bla OXA-232. The 50514 bp bla OXA-181-harbouring plasmid, pOXA-181_YML0508, was X3-type with a conjugation frequency to Escherichia coli of 1.94×10−4 transconjugants per donor. The bla OXA-232 gene was located on a 6141 bp ColKP3-type plasmid, pOXA-232_WSD, that was identical in the ST76 and ST15 K. pneumoniae isolates. This plasmid could be transferred from K. pneumoniae to E. coli at low frequency, 8.13×10−6 transconjugants per donor. Comparative analysis revealed that the X3 plasmid acquired the bla OXA-48-like gene via IS3000-mediated co-integration of the ColKP3-type plasmid. Our study highlights how plasmid integration and rearrangements can contribute to the spread of bla OXA-48-like genes, which provides important clues for clinical prevention of the dissemination of K. pneumoniae strains carrying bla OXA-48-like carbapenemases.
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Diazepam’s antifungal activity in fluconazole-resistant Candida spp. and biofilm inhibition in C. albicans: evaluation of the relationship with the proteins ALS3 and SAP5
Lisandra Juvêncio da Silva, Fátima Daiana Dias Barroso, Lucas Sousa Vieira, Daniel Roberto Carlos Mota, Bruna Kelly da Silva Firmino, Cecília Rocha da Silva, Vitória Pessoa de Farias Cabral, Thiago Mesquita Cândido, Lívia Gurgel do Amaral Valente Sá, Wildson Max Barbosa da Silva, Jacilene Silva, Emmanuel Silva Marinho, Bruno Coelho Cavalcanti, Manoel Odorico de Moraes, Hélio Vitoriano Nobre Júnior and João Batista de Andrade NetoThe genus Candida spp. has been highlighted as one of the main etiological agents causing fungal infections, with Candida albicans being the most prominent, responsible for most cases of candidemia. Due to its capacity for invasion and tissue adhesion, it is associated with the formation of biofilms, mainly in the environment and hospital devices, decreasing the effectiveness of available treatments. The repositioning of drugs, which is characterized by the use of drugs already on the market for other purposes, together with molecular-docking methods can be used aiming at the faster development of new antifungals to combat micro-organisms. This study aimed to evaluate the antifungal effect of diazepam on mature C. albicans biofilms in vitro and its action on biofilm in formation, as well as its mechanism of action and interaction with structures related to the adhesion of C. albicans, ALS3 and SAP5. To determine the MIC, the broth microdilution test was used according to protocol M27-A3 (CLSI, 2008). In vitro biofilm formation tests were performed using 96-well plates, followed by molecular-docking protocols to analyse the binding agent interaction with ALS3 and SAP5 targets. The results indicate that diazepam has antimicrobial activity against planktonic cells of Candida spp. and C. albicans biofilms, interacting with important virulence factors related to biofilm formation (ALS3 and SAP5). In addition, treatment with diazepam triggered a series of events in C. albicans cells, such as loss of membrane integrity, mitochondrial depolarization and increased production of EROs, causing DNA damage and consequent cell apoptosis.
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Rifampin-resistance-associated mutations in the rifampin-resistance-determining region of the rpoB gene of Mycobacterium tuberculosis clinical isolates in Shanghai, PR China
Introduction. Resistance to rifampin (RIF) in Mycobacterium tuberculosis infection is associated with mutations in the rpoB gene coding for the β-subunit of RNA polymerase. The contribution of various rpoB mutations to the development and level of RIF resistance remains elusive.
Hypothesis/Gap Statement. Various rpoB mutations may be associated with differential levels of RIF resistance.
Aim. This study aimed to investigate the relationship between specific rpoB mutations and the MICs of RIF and rifabutin (RFB) against M. tuberculosis .
Methodology. Of the 195 clinical isolates, 105 and 90 isolates were randomly selected from isolates resistant to RIF and sensitive to RIF, respectively. The MICs of 12 agents for M. tuberculosis isolates were determined using commercial Sensititre M. tuberculosis MIC plates and the broth microdilution method. Strains were screened for rpoB mutations by DNA extraction, rpoB gene amplification and DNA sequence analysis.
Results. One hundred isolates (95.24 %) were found to have mutations in the RIF-resistance-determining region (RRDR) of the rpoB gene. Three rpoB mutations were identified in 90 RIF-susceptible isolates. Out of 105 isolates, 86 (81.90 %) were cross-resistant to both RIF and RFB. The most frequent mutation occurred at codons 450 and 445. We also found a novel nine-nucleotide (ATCATGCAT) deletion (between positions 1543 and 1551) in the rpoB gene in two strains (1.90 %) with resistance to RIF, but susceptibility to RFB. In addition, the mutation frequency at codon 450 was significantly higher in RIF-resistant/RFB-resistant (RIFR/RFBR) strains than in RIFR/RFBS strains (75.58 % versus 21.05 %, P<0.01), whereas the mutation frequency at codon 435 was significantly lower in RIFR/RFBR strains than in RIFR/RFBS strains (1.16 % versus 26.32 %, P<0.01).
Conclusion. Our data support previous findings, which reported that various rpoB mutations are associated with differential levels of RIF resistance. The specific mutations in the rpoB gene in RIFR/RFBR isolates differed from those in the RIFR/RFBS isolates. A novel deletion mutation in the RRDR might be associated with resistance to RIF, but not to RFB. Further clinical studies are required to investigate the efficacy of RFB in the treatment of infections caused by M. tuberculosis strains harbouring these mutations.
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Co-infections of two carbapenemase-producing Enterobacter hormaechei clinical strains isolated from the same diabetes individual in China
Introduction. Since mcr-1 was first reported in China, there have been ten variants of MCR appearing nationwide so far. Multidrug-resistant Enterobacteriaceae bacteria carrying both NDM and MCR have become a serious threat to global public health.
Hypothesis/Gap Statement. The genetic structure of mcr-9 needs to be better understood in order to better prevent and control the transmission of drug-resistant genes.
Aims. The aim of this study was to characterize the presence of two Enterobacter hormaechei isolates, which carries bla NDM-5 CME2 and the coexistence of mcr-9 and bla NDM-1 strain CMD2, which were isolated from a patient with diabetes in Sichuan, China.
Methodology. The microbroth dilution method was used for antibiotic susceptibility. Conjugation experiment was used to investigate the transferability of bla NDM-1, bla NDM-5 and mcr-9. Whole-genome sequencing was performed on Illumina HiSeq platform. The ability of biofilm formation was detected by crystal-violet staining, the virulence of the bacteria was measured by Galleria mellonella killing assay.
Results. bla NDM-5 carrier CME2 and CMD2 with bla NDM-1 and mcr-9 were resistant to carbapenems, β-lactam, aminoglycoside, quinolone and tetracycline, while CMD2 was also resistant to colistin. Conjugation assay and plasmid replicon typing further demonstrated that both bla NDM-1 and bla NDM-5 were respectively present on the self-transferrable IncX3 plasmid, mcr-9 was located on the self-transferrable IncHI2 plasmid. Through the analysis of mcr-9 gene context, the structure was DUF4942-rcnR-rcnA-copS-IS903-mcr-9-wbuC-qseC-qseB-IS1R-ΔsilR-IS903, bla NDM-1 context was IS3000-ΔISAba125-IS5-bla NDM-1-ble-trpF-groS-groL-insE-ΔIS26 structure, bla NDM-5 structure was IS3000-bla NDM-5-ble-trpF-dsbC-ΔIS26-umuD-ISKox3-tnpR-parA. Biofilm formation of CME2 was stronger than CMD2. There was no significant difference in virulence between the two strains.
Conclusion. This study reveals two multiple drug-resistant E. hormaechei isolates from diabetes patient samples. E. hormaechei carrying two NDM-resistant genes is already a serious threat, where MCR is an important cause of treatment failure in bacterial infections. This study is a reminder not only to prevent infection in patients with diabetes, but also to constantly monitor the epidemic and spread of the drug-resistant gene.
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Emergence of a plasmid-borne tigecycline resistance in Klebsiella pneumoniae in Vietnam
More LessTigecycline is a last-resort antimicrobial used to treat multidrug-resistant Gram-negative bacterial infections. One of the common antimicrobial resistance mechanisms is the efflux pump system composed of membrane protein complexes to excrete xenobiotic substrates. Recently, a novel gene cluster, tmexCD1-toprJ1, encoding the resistance–nodulation–cell division (RND) efflux pump was identified on plasmids in Klebsiella pneumoniae isolates in China. TMexCD1-TOprJ1 was found to be capable of excreting multiple antimicrobials, including tigecycline, which contributed to the strain's resistance. In this study, we identified K. pneumoniae isolates harbouring the tmexCD1-toprJ1 genes outside of China for the first time. Two tigecycline-resistant K. pneumoniae isolates belonging to ST273 by multilocus sequence typing were collected from different patients in a medical institution in Hanoi, Vietnam, in 2015. Whole-genome sequence analysis revealed that these isolates harboured a 288.0 kb tmexCD1-toprJ1–carrying plasmid with IncFIB and IncHI1B replicons. The tmexCD1-toprJ1 gene cluster was surrounded by several mobile gene elements, including IS26, and the plasmids had high sequence identity with that of K. pneumoniae isolated in China. Our finding suggests that the horizontal spread of tigecycline resistance mediated by tmexCD1-toprJ1–carrying plasmids has occurred in Vietnam and other countries, and raises concern about the further global dissemination.
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Fidaxomicin has high in vitro activity against Mycobacterium tuberculosis
More LessThis study aimed to evaluate whether the antibiotic fidaxomicin has in vitro activity against Mycobacterium tuberculosis (Mtb). 38 fully drug-sensitive Mtb strains and 34 multidrug-resistant tuberculosis (MDR-TB) strains were tested using the microplate alamar blue assay (MABA) method to determine the minimum inhibitory concentrations (MICs) for fidaxomicin and rifampicin. Fidaxomicin has high in vitro activity against Mtb and is a potential drug to treat Mtb, and MDR-TB infections in particular.
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Comparison of drug-susceptibility patterns and gene sequences associated with clarithromycin and azithromycin resistance in Mycobacterium abscessus complex isolates and evaluation of the accumulation of intrinsic macrolide resistance
More LessIntroduction. Mycobacterium abscessus complex (MABC) is an infectious agent associated with macrolide resistance and treatment failure.
Hypothesis/Gap Statement. Despite drug-susceptibility testing for MABC isolates including clarithromycin (CAM), long-term treatment with azithromycin (AZM) for MABC disease is recommended.
Aim. We compared phenotypic and genotypic resistance to AZM and CAM in clinical isolates and evaluated the accumulation of intrinsic macrolide resistance (AIM) and morphological changes by macrolides exposure.
Methodology. Forty-nine isolates were characterized regarding erm(41) sequevars. Sequencing data were compared to the nucleotide sequence of rrl and whiB7. The AIM MIC was performed in three reference strains and 15 isolates were randomized [each set of five isolates with M. abscessus subsp. abscessus (MAA) T28, MAA C28 and subsp. massiliense (MAM)].
Results. The 49 isolates were distributed as 24 MAA T28, 5 MAA C28 and 20 MAM. The MIC50 values to CAM at day 3 in MAA T28, C28 and MAM were 1, 0.12 and 0.12 µg ml−1, while those at day 14 were 32, 0.5 and 0.12 µg ml−1, respectively. The AZM-MIC50 values at day 3 of the above isolates were 4, 0.25 and 0.5 µg ml−1, while those at day 14 were >64, 0.5 and 0.5 µg ml−1, respectively. Neither mutations in rrl of MAA T28 with acquired resistance nor deletions in whiB7 of MAA T28 without inducible resistance were observed . For AIM MIC, MAA T28 showed that the time-to-detection of AZM resistance was significantly faster over that of CAM (P<0.05). Morphological changes were not determined in all isolates.
Conclusion. Our findings did not support the suggestion for the preferential use of AZM for, at least, MAA T28 disease due to the high-level MIC value and the increased AIM. The long duration of AZM-based treatment eventually may favour the emergence of isolates with a high-level of intrinsic resistance.
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Comparative study of multidrug-resistant Enterococcus faecium obtained from different hosts
Introduction. The possible transfer of antimicrobial resistance genes between Enterococcus faecium isolates from humans and different animal species, including those not covered by monitoring programs (e.g. pet and wildlife), poses a serious threat to public health.
Hypothesis/Gap Statement. Little is known about occurrence and mechanisms of phenomenon of multidrug resistance of E. faecium isolated from various host species in Poland.
Aim. The aim of the study was to characterize multidrug-resistant E. faecium isolated from humans and animals (livestock, pets and wildlife) in terms of the occurrence of genetic markers determining resistance.
Methodology. Bacterial isolates were tested for phenotypic resistance and the presence of genes encoding resistance to macrolides, tetracycline, aminoglycosides, aminocyclitols and phenicols as well as efflux pump (emeA), resolvase (tndX) and integrase (Int-Tn) genes. The quinolone resistance-determining regions of gyrA and parC were sequenced.
Results. Human isolates of E. faecium were characterized by high-level resistance to: ciprofloxacin, enrofloxacin, erythromycin (100 %), as well, as aminoglycosides resistance (kanamycin – 100%, streptomycin – 78 %, gentamicin – 78%). Regardless of the animal species, high level of resistance of E. faecium to tetracycline (from 88–100 %), erythromycin (from 82–94 %) and kanamycin (from 36–100 %) was observed. All E. faecium isolates from wildlife were resistant to fluoroquinolones. However, full susceptibility to vancomycin was observed in all isolates tested. Phenotypic antimicrobial resistance of E. faecium was identified in the presence of the following resistance genes: erm(B) (70%), msr(A) (50 %), tet(L) (35 %), tet(K) (34 %), tet(M) (76 %), aac(6’)-Ie-aph(2″)-Ia (25%), ant(6)-Ia (31%), aph(3)-IIIa (68 %), (tndX) (23 %), and integrase gene (Int-Tn) (34 %). A correlation between an amino acid substitution at positions 83 and 87 of gyrA and position 80 of parC and the high-level fluoroquinolone resistance in E. faecium has been observed as well.
Conclusion. The level and range of antimicrobial resistance and the panel of resistance determinants is comparable between E. faecium isolates, despite host species.
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- Clinical Microbiology
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The first UK report of a rare Cryptosporidium hominis genetic variant isolated during a complex Scottish swimming pool outbreak
More LessCryptosporidium species are responsible for causing the majority of parasite-related gastrointestinal infections in the UK. This report describes an outbreak of 12 laboratory-confirmed cryptosporidiosis cases identified as part of a Scottish swimming pool investigation, with 9 primary and 3 secondary cases occurring over an 8-week period. Molecular speciation was successful for 11/12 cases, which revealed 10 Cryptosporidium hominis cases and 1 Cryptosporidium parvum case. Of the 10 C. hominis cases, further typing identified 7 as being an unusual sub-type, IbA6G3, which is the first description in the UK of this rare variant. The remaining three C. hominis cases were identified as the common IbA10G2 subtype. Following implementation of control measures on two occasions, no further cases were reported. This report highlights the importance of molecular typing to identify and characterize outbreaks, and emphasizes the need to adhere to swimming pool guidance. It also raises awareness of the potential for outbreaks to involve multiple species/sub-types, and emphasizes the importance of strong public health leadership to ensure effective multi-agency investigations and management of outbreaks.
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Quality indicators for blood culture: 1 year of monitoring with BacT/Alert Virtuo at a French hospital
More LessIntroduction. Blood culture (BC) remains the gold standard for the diagnosis of bloodstream infection. Clinical microbiology laboratories must ensure the quality of their BC process from receipt to definitive results.
Aim. In this study, we followed the evolution of different quality indicators for BCs over the first year of implementation of the BacT/Alert Virtuo system in a French hospital.
Methodology. In our laboratory, we instituted regular monitoring of several quality indicators to track (i) delays in sample registration, (ii) delays in loading BC bottles in our incubating system (BacT/Alert Virtuo) after registration, (iii) the volume of blood in bottles and (iv) the contamination rates.
Results. For 53 892 BC bottles loaded in the BacT/Alert Virtuo from 23 January to 31 December 2019, the delays in sample registration, loading and unloading were respectively 3.5 h±0.016, 44 min±0.209 and 5.8 h±0.0727. Intriguingly, the automated process performed by the BacT/Alert Virtuo system to check the blood volume in bottles was only performed for 60 % of the loaded bottles. Among these, 30 % contained the recommended volume of blood (between 7 and 13 ml). Finally, the contamination rate was found to be 27.2 % for samples at our institution.
Conclusions. The delays in sample registration, loading and unloading were found to be acceptable, even though they could be improved by ensuring a continuous service during the night duty period. Furthermore, the percentage of volumes measured is insufficient and must be improved and the majority of bottles do not contain the recommended blood volume.
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Microbiological pattern of prosthetic hip and knee infections: a high-volume, single-centre experience in China
Yali Yu, Yiyi Kong, Jing Ye, Aiguo Wang and Wenteng SiIntroduction. Prosthetic joint infection (PJI) is a serious complication after arthroplasty, which results in high morbidity, prolonged treatment and considerable healthcare expenses in the absence of accurate diagnosis. In China, microbiological data on PJIs are still scarce.
Hypothesis/Gap Statement. The incidence of PJI is increasing year by year, and the proportion of drug-resistant bacteria infection is nicreasing, which brings severe challenges to the treatment of infection.
Aim. This study aimed to identify the pathogens in PJIs, multi-drug resistance, and evaluate the effect of the treatment regimen in patients with PJI.
Methodology. A total of 366 consecutive cases of PJI in the hip or knee joint were admitted at the Orthopedic Surgery Center in Zhengzhou, China from January 2012 to December 2018. Infections were confirmed in accordance with the Infectious Diseases Society of America and the Musculoskeletal Infection Society (MSIS) criteria. Concurrently, patient demographic data, incidence and antibiotic resistance were investigated. Statistical differences were analysed using Fisher’s exact test or chi-square test.
Results. Altogether, 318 PJI cases satisfying the inclusion criteria were enrolled in this study, including 148 with hip PJIs and 170 with knee PJIs. The average age of patients with hip PJIs was lesser than that of patients with knee PJIs (56.4 vs. 68.6 years). Meanwhile, coagulase-negative staphylococcus (CNS, n=81, 25.5 %) was the predominant causative pathogen, followed by Staphylococcus aureus (n=67, 21.1 %). Methicillin-resistant Staphylococcus (MRS) was identified in 28.9 % of PJI patients. In addition, fungus accounted for 4.8 % (n=15), non-tuberculosis mycobacterium accounted for 1.6 % (n=5), polymicrobial pathogens accounted for 21.7 % (n=69), and Gram-negative bacteria accounted for 7.9 % (n=25) of the total infections. The results of antibiotic susceptibility testing showed that gentamicin and clindamycin β-lactam antibiotics were poorly susceptible to Gram-positive isolates, but they were sensitive to rifampicin, linezolid and vancomycin. While antibiotics such as amikacin and imipenem were effective against Gram-negative bacteria, there was a high resistance rate of other pathogens to gentamicin, clindamycin and some quinolone antibacterial drugs. Empirical antibiotic treatment should combine vancomycin and cephalosporin, levofloxacin or clindamycin. When the pathogen is confirmed, the treatment should be individualized.
Conclusions. The prevalence of culture-negative PJIs is still very high. Gram-positive bacteria are still the main type of pathogens that cause PJIs. Attention should be paid to the high incidence of MRS, such as MRSA and MR-CNS, among PJI patients. Empirical antibiotic treatment should cover Gram-positive isolates, especially Staphylococcus .
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Phenotypic and genetic characteristics of Streptococcus mutans isolates from site-specific dental plaque in China
Introduction. Streptococcus mutans is an important cariogenic microbe.
Hypothesis/Gap Statement. The potential characteristics of S. mutans isolates from site-specific dental plaque are still not clear.
Aim. This study aimed to investigate the phenotypic and genetic characteristics of S. mutans isolates from site-specific dental plaque in China.
Methodology. We used S. mutans isolated from children with early-childhood caries (ECC) and caries-free children to compare the phenotypic and genetic characteristics of S. mutans from site-specific dental plaque samples. The ECC subjects presented two sites: a cavitated lesion and a sound surface. The caries-free subjects presented one sound surface. Growth pattern, biofilm, decrease in pH, extracellular polysaccharide, expression levels of virulence-related genes, multilocus sequence typing (MLST) and phylogenetic trees were evaluated among these three sites.
Results. The phenotypes detected between the cavitated and sound surfaces of ECC children were similar. However, the capacity for biofilm formation, pH drop and expression levels of genes (gtfB and spaP) of S. mutans in the caries-free group were lower compared with those of the ECC group. We identified 44 new alleles and 77 new sequence types. More than 90 % of the children with ECC shared an identical sequence type. The distribution of sequence types among different subjects showed diversity, and child-to-child transmission was detected.
Conclusions. This is the first report of MLST on site-specific dental plaques in a single subject, and indicates that S. mutans isolated from site-specific dental plaque of a single subject showed similar phenotypes as a result of the isolates were closely related.
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Comparative genome analysis of three Group A Streptococcus dysgalactiae subsp. equisimilis strains isolated in Japan
Introduction . Streptococcus dysgalactiae subsp. equisimilis (SDSE) is a β-hemolytic streptococcus that causes severe invasive streptococcal infections, especially in the elderly and people with underlying diseases. SDSE strains are primarily characterized by Lancefield group G or C antigens.
Hypothesis/Gap Statement. We have previously reported the prevalence of Lancefield group A SDSE (GA-SDSE) strains in Japan and have analysed the draft genome sequences of these strains. As GA-SDSE is a rare type of SDSE, only one complete genome has been sequenced to date.
Aim. The present study is focused on genetic characteristics of GA-SDSE strains. In order to examine molecular characteristics, we also tested growth inhibition of other streptococci by GA-SDSE.
Methodology. We determined the complete genome sequences of three GA-SDSE strains by two new generation sequencing systems (short-read and long-read sequencing data). Using the sequences, we also conducted a comparative analysis of GA-SDSE and group C/G SDSE strains. In addition, we tested multiplex and quantitative PCRs targeting the GA-SDSE, group G SDSE, and S. pyogenes .
Results. We found a group-specific conserved region in GA-SDSE strains that is composed of genes encoding predicted anti-bacteriocin and streptococcal lantibiotic (Sal) proteins. Multiplex and quantitative PCRs targeting the GA-SDSE-specific region were able to distinguish between GA-SDSE, other SDSE, and S. pyogenes strains. The growth of GA-SDSE was suppressed in the presence of group G SDSE, indicating a possible explanation for the low frequency of isolation of GA-SDSE.
Conclusion. The comparative genome analysis shows that the genome of GA-SDSE has a distinct arrangement, enabling the differentiation between S. pyogenes , GA-SDSE, and other SDSE strains using our PCR methods.
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The immunomodulatory activity of secnidazole–nitazoxanide in a murine cryptosporidiosis model
More LessIntroduction. Cryptosporidium parvum causes intestinal parasitic infections affecting both immunosuppressed and immunocompetent individuals.
Gap statement. Given the absence of effective treatments for cryptosporidiosis, especially in immunodeficient patients, the present study was designed to assess the therapeutic efficacy of secnidazole (SEC) and its combination with nitazoxanide (NTZ) in comparison to single NTZ treatment in relation to the immune status of a murine model of C. parvum infection.
Methodology. The infected groups were administered NTZ, SEC or NTZ–SEC for three or five successive doses. At days 10 and 12 post-infection (p.i.), the mice were sacrificed, and the efficacy of the applied drugs was evaluated by comparing the histopathological alterations in ileum and measuring the T helper Th1 (interferon gamma; IFN-γ), Th2 [interleukin (IL)-4 and IL-10] and Th17 (IL-17) cytokine profiles in serum.
Results. The NTZ–SEC combination recorded the maximal reduction of C. parvum oocyst shedding, endogenous stages count and intestinal histopathology, regardless of the immune status of the infected mice. The efficacy of NTZ–SEC was dependent on the period of administration, as the 5 day-based treatment protocol was also more effective than the 3 day-based one in terms of immunocompetence and immunosuppression. The present treatment schedule induced an immunomodulatory effect from SEC that developed a protective immune response against C. parvum infection with reduced production of serum IL-17, IFN-γ, IL-4 and IL-10.
Conclusions. Application of NTZ–SEC combined therapy may be useful in treatment of C. parvum, especially in cases involving immunosuppression.
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Clinical relevance and antimicrobial susceptibility profile of the unknown human pathogen Corynebacterium aurimucosum
Introduction. Even though Corynebacterium aurimucosum has been described in 2002, this species has long been underestimated due to the unreliability of conventional identification methods and only a few cases of infections have been reported.
Hypothesis/Gap Statement. Little is known about clinical significance and antimicrobial susceptibility profile of this uncommon species.
Aim. To evaluate the clinical relevance of C. aurimucosum and its antimicrobial susceptibility profile.
Methodology. All C. aurimucosum isolates, collected from 2010 to 2019 in 10 French university hospitals, were retrospectively included. Demographic, clinical and microbiological data were collected for all cases. Antimicrobial susceptibility testing was performed according to the 2019 EUCAST guidelines.
Results. Fifty-seven clinical isolates of C. aurimucosum were collected in 57 patients (median age, 65.8 years; male/female sex ratio, 1.1), mostly from urine (28 %), blood culture (28 %) and bone/synovial fluid (19 %) samples. Of them, 14 cases of infection were confirmed, mainly bone and joint infections (50 %) followed by urinary tract infections (UTIs) (21 %), bacteremia (14 %), skin and soft-tissue infections (14 %). C. aurimucosum was recovered in pure culture in 36 % of cases (UTIs and bacteremia) while mixed cultures were observed for other infections. By testing 52 clinical isolates in vitro, this species appeared to be fully susceptible to linezolid and vancomycin while most isolates (>80 %) were susceptible to amoxicillin (MIC90, 2 µg ml−1), gentamicin, tetracycline and rifampicin. Both cefotaxime and ciprofloxacin seemed to have a limited activity (ca. 50 % of susceptible strains). The MIC distribution for ciprofloxacin showed a bimodal profile with a population of highly-resistant strains with MICs >2 µg ml−1. Most isolates (>90 %) were categorized as resistant to penicillin G and clindamycin.
Conclusion. C. aurimucosum should be considered as an actual opportunistic pathogen, and treatment with amoxicillin, vancomycin or linezolid should be preferred.
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Profile of specific antibodies to the SARS-CoV-2
In this work, we studied the profile of IgM and IgG antibody responses to SARS-CoV-2 in 32 patients with COVID-19 from day 1 to day 24. IgM remained measurable for a much shorter period than IgG, suggesting that IgG antibody may represent the primary immune response.
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- Disease, Diagnosis and Diagnostics
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Detection of parC gene mutations associated with quinolone resistance in Mycoplasma genitalium: evaluation of a multiplex real-time PCR assay
Introduction. Increasing levels of antibiotic resistance are complicating treatment for the sexually transmitted pathogen Mycoplasma genitalium . Resistance to fluoroquinolones is associated with mutations in the parC gene. Although the precise mutations conferring resistance are not fully understood, the single nucleotide polymorphism (SNP) G248T/S83I is most implicated.
Aim. To evaluate the performance of the MG+parC(beta2) assay (SpeeDx, Australia), which detects single nucleotide polymorphisms (SNPs) in the parC gene at amino acid position S83 (A247C/S83R, G248T/S83I, G248A/S83N) and D87 (G259A/D87N, G259T/D87Y, G259C/D87H).
Methods. Clinical samples were analysed by MG+parC(beta2) assay and results compared to Sanger sequencing. Sensitivity, specificity, and predictive value for treatment failure were calculated.
Results. From analysis of 205 samples, the MG+parC(beta2) assay performed with a high sensitivity 98.2% (95% CI:90.3–100) and specificity 99.3% (95% CI:96.3–100) for parC SNP detection with a kappa of 0.97 (95% CI:0.94–1.00). The predictive value of G248T/S83I detection (the most common SNP, prevalence of 13% in the study population) was analysed with respect to treatment failure (patients received sequential doxycycline-moxifloxacin). The positive-predictive-value for moxifloxacin failure after detection of S83I was only 44% (95% CI:24.4–65.1), while negative-predictive-value was high at 96.9% (95% CI:92.7–99.0), suggesting that other SNPs are contributing to resistance.
Conclusion. MG+parC(beta2) performed with high concordance compared to Sanger sequencing. Such qPCR assays can assist in understanding causes of treatment failure, inform the development of diagnostic assays, and can be applied to surveillance of mutations in populations. Due to an incomplete understanding of the basis for fluoroquinolone resistance, such tests do not appear to be ready for clinical application.
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Comparative effects of viral-transport-medium heat inactivation upon downstream SARS-CoV-2 detection in patient samples
Jamie L. Thompson, Angela Downie Ruiz Velasco, Alice Cardall, Rebecca Tarbox, Jaineeta Richardson, Gemma Clarke, Michelle Lister, Hannah C. Howson-Wells, Vicki M. Fleming, Manjinder Khakh, Tim Sloan, Nichola Duckworth, Sarah Walsh, Chris Denning, C. Patrick McClure, Andrew V. Benest and Claire H. SeedhouseIntroduction. The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At the time of writing, positive detection of the SARS-CoV-2 virus remains the only method for diagnosing COVID-19 infection. Rapid upscaling of national SARS-CoV-2 genome testing presented challenges: (1) Unpredictable supply chains of reagents and kits for virus inactivation, RNA extraction and PCR-detection of viral genomes. (2) Rapid time to result of <24 h is required in order to facilitate timely infection control measures.
Hypothesis. Extraction-free sample processing would impact commercially available SARS-CoV-2 genome detection methods.
Aim. We evaluated whether alternative commercially available kits provided sensitivity and accuracy of SARS-CoV-2 genome detection comparable to those used by regional National Healthcare Services (NHS).
Methodology. We tested several detection methods and tested whether detection was altered by heat inactivation, an approach for rapid one-step viral inactivation and RNA extraction without chemicals or kits.
Results. Using purified RNA, we found the CerTest VIASURE kit to be comparable to the Altona RealStar system currently in use, and further showed that both diagnostic kits performed similarly in the BioRad CFX96 and Roche LightCycler 480 II machines. Additionally, both kits were comparable to a third alternative using a combination of Quantabio qScript one-step Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) mix and Centre for Disease Control and Prevention (CDC)-accredited N1 and N2 primer/probes when looking specifically at borderline samples. Importantly, when using the kits in an extraction-free protocol, following heat inactivation, we saw differing results, with the combined Quantabio-CDC assay showing superior accuracy and sensitivity. In particular, detection using the CDC N2 probe following the extraction-free protocol was highly correlated to results generated with the same probe following RNA extraction and reported clinically (n=127; R2=0.9259).
Conclusion. Our results demonstrate that sample treatment can greatly affect the downstream performance of SARS-CoV-2 diagnostic kits, with varying impact depending on the kit. We also showed that one-step heat-inactivation methods could reduce time from swab receipt to outcome of test result. Combined, these findings present alternatives to the protocols in use and can serve to alleviate any arising supply-chain issues at different points in the workflow, whilst accelerating testing, and reducing cost and environmental impact.
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Helicobacter suis and Helicobacter pylori infection in a colony of research macaques: characterization and clinical correlates
Introduction. Helicobacter suis ( Helicobacter heilmannii type 1) commonly infects nonhuman primates but its clinical importance is in question.
Aim. To characterize H. suis infection in a colony of rhesus macaques (Macaca mulatta) used in cognitive neuroscience research.
Hypothesis/Gap Statement. Inquiries into the nature of Helicobacter suis in nonhuman primates are required to further define the organism’s virulence and the experimental animal’s gastric microbiome.
Methodology. Animals with and without clinical signs of vomiting and abdominal pain (n=5 and n=16, respectively) were evaluated by histology, culture, PCR amplification and sequencing, fluorescent in situ hybridization (FISH) and serology. Three of the five animals with clinical signs, an index case and two others, were evaluated before and after antimicrobial therapy.
Results. The index animal had endoscopically visible ulcers and multifocal, moderate, chronic lymphoplasmacytic gastritis with intraglandular and luminal spiral bacteria. Antimicrobial therapy in the index animal achieved histologic improvement, elimination of endoscopically visible ulcers, and evident eradication but clinical signs persisted. In the other treated animals, gastritis scores were not consistently altered, gastric bacteria persisted, but vomiting and abdominal discomfort abated.
Nineteen of 21 animals were PCR positive for H. suis and five animals were also PCR positive for H. pylori . Organisms were detected by FISH in 17 of 21 animals: 16S rRNA sequences of two of these were shown to be H. suis . Mild to moderate lymphoplasmacytic gastritis was seen in antrum, body and cardia, with antral gastritis more likely to be moderate than that of the body.
Conclusion. No clear association between the bacterial numbers of Helicobacter spp. and the degree of inflammation was observed. H. suis is prevalent in this colony of Macaca mulatta but its clinical importance remains unclear. This study corroborates many of the findings in earlier studies of H. suis infection in macaques but also identifies at least one animal in which gastritis and endoscopically visible gastric ulcers were strongly associated with H. suis infection. In this study, serology was an inadequate biomarker for endoscopic evaluation in diagnosis of H. suis infection.
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Surveillance of invasive meningococcal disease in the south of Brazil: considerations of immunization programme
THIS ARTICLE HAS BEEN RETRACTED
Invasive meningococcal disease (IMD) is a major cause of meningitis and septicaemia worldwide. The switches in serogroup predominance contribute to the unpredictable nature of the disease with significant health impacts. The aim of this study was to determine the epidemiological profile of IMD in Rio Grande do Sul, Santa Catarina and Paraná, three states in the south of Brazil. All meningitis cases confirmed by clinical and/or laboratory criteria notified to the national information system for notifiable diseases between 2015 and 2019 were analysed. Proportions of serogroup and incidence by age were calculated. A total of 17 894 cases of IMD were reported during this period. Of these, 9029 cases (50 %) were due to serogroup C. Furthermore, serogroup W was responsible for almost half of the cases among children younger than 5 years old during 2017 and 2018, with an overall incidence of 33.3 cases per 100 000 infants. Despite the reduction in serogroup C after the introduction of meningococcal C conjugate vaccine into a childhood immunization programme in Brazil, it remains a significant healthcare issue in the south of the country. Changes in disease epidemiology were observed and serogroup W was the most common among children below 5 years of age in 2017 and 2018. Although future cost-effectiveness studies are necessary, our results could have future implications for meningococcal vaccination programmes.
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The simple direct slide method is comparable to indirect Lowenstein Jensen proportion culture for detecting rifampicin resistant tuberculosis
Introduction. Drug resistant tuberculosis remains a worldwide problem that requires prompt diagnosis.
Hypothesis/Gap statement. The WHO recommended direct, rapid Xpert MTB/RIF is prohibitively costly, therefore, there is a need to validate a rapid, affordable DST for use in low- and middle-income settings.
Aim. The technical performance and time to results of a simple, direct microscopy-based slide DST (SDST) assay for diagnosis of rifampicin-resistant TB was evaluated in Uganda.
Methodology. Sputum samples from 122 smear-positive re-treatment TB patients presenting to the TB treatment centre at Uganda’s National Referral Hospital, Mulago, Kampala, Uganda were examined. The sputum samples were tested by the direct SDST which was compared to the indirect Lowenstein Jensen Proportion Method (LJDST) method as the gold standard. The time to results was defined as the time from DST setting to results interpretation. The results were further analysed for sensitivity and specificity as well as agreement between LJDST and SDST for rifampicin resistance determination.
Results. A total of 117 smear positive sputum samples with valid results for both tests were compared. The median time to results for SDST was 14 days with an interquartile range (IQR) of 10–14 days compared to 60 days with IQR of 60–75 days for LJDST. The number for rifampicin resistance by the gold standard LJDST was 26. The SDST had a sensitivity of 96 % (95 %; CI 81–99 %) and a specificity of 97.8 % (95 %; CI 93–100 %). The Positive Predictive and Negative Predictive values for SDST were 92.3 % (95 %; CI 76.8–99 %) and 98.9 % (95 %; CI 94–100 %), respectively. The kappa agreement between SDST and LJDST was 92.3 %.
Conclusion. The SDST was found to be a rapid and accurate direct test for the detection of rifampicin resistance among retreatment TB cases in low-income settings.
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Genetic relatedness of Streptococcus dysgalactiae isolates causing recurrent bacteraemia
More LessIntroduction. Streptococcus dysgalactiae subspecies equisimilis (SDSE) is becoming increasingly recognized as an important human pathogen. Recurrent bacteremia with SDSE has been described previously.
Aim. The aims of the study were to establish the genetic relatedness of SDSE isolates with emm-type stG643 that had caused recurrent bacteraemia in three patients and to search for signs of horizontal gene transfer of the emm gene in a collection of SDSE stG643 genomes.
Hypothesis. Recurring SDSE bacteremia is caused by the same clone in one patient.
Methodology. Whole genome sequencing of 22 clinical SDSE stG643 isolates was performed, including three paired blood culture isolates and sixteen isolates from various sites. All assemblies were aligned to a reference assembly and SNPs were extracted. A total of 53 SDSE genomes were downloaded from GenBank. Two phylogenetic trees, including all 75 SDSE isolates, were created. One tree was based on the emm gene only and one tree was based on all variable positions in the genomes.
Results. The genomes from the three pairs of SDSE isolates showed high sequence similarity (1–17 SNPs difference between the pairs), whereas the median SNP difference between the 22 isolates in our collection was 1694 (range 1–11257). The paired isolates were retrieved with 7–53 months between episodes. The 22 SDSE isolates from our collection formed a cluster in the phylogenetic tree based on the emm gene, while they were more scattered in the tree based on all variable positions.
Conclusions. Our results show that the paired isolates were of the same clonal origin, which in turn supports carriage between bacteraemia episodes. The phylogenetic analysis indicates that horizontal gene transfer of the emm-gene between some of the SDSE isolates has occurred.
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Evaluation of true point of care molecular assay using fingerstick capillary whole blood for diagnosis of hepatitis C infection
More LessAt present, the available point of care (POC) molecular assays for hepatitis C are not considered as true POC due to sample collection and processing requiring minimal laboratory infrastructure. A new POC Xpert HCV VL Fingerstick (Xpert FS) precludes such requirements where specimen collected by simple fingerstick can be loaded directly into the test cartridge with results available within 60 min. The present study compared the performance of this assay for HCV RNA quantitation using both capillary whole blood (CWB) and venous whole blood (VWB) with plasma HCV RNA performed on Abbott Real Time HCV PCR. CWB via fingerstick and VWB via venipuncture collected from serologically confirmed HCV-infected participants were loaded into Xpert HCV VL WB for viral load estimation. Simultaneously Abbott Real Time HCV PCR assay was also performed using plasma (reference method). Among the enrolled participants (n=157), the mean age was 46.22±14.79 years and 63 % were male. HCV RNA was detected in 100 cases (63.7 %), median 5.69 (IQR: 5.00–6.32)log10IU ml−1 on the reference method. Xpert FS showed 100 % sensitivity and specificity using both CWB and VWB. The median viral loads detected in CWB and VWB were 5.52 (IQR: 4.59–6.15) and 5.48 (IQR: 4.61–6.07)log10IU ml−1, respectively. Xpert FS offers potential as true POC enabling accurate diagnosis in a single patient visit to the health-care facility, hence may reduce the number of dropouts with a confirmed diagnosis. However, further real-time studies with larger sample size are warranted.
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- Medical Mycology
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Implication of efflux pumps and ERG11 genes in resistance of clinical Trichosporon asahii isolates to fluconazole
S. Abbes, H. Sellami, S. Neji, H. Trabelsi, F. Makni and A. AyadiIntroduction. Trichosporon asahii has been recognized as an opportunistic agent having a limited sensitivity to antifungal treatment.
Hypothesis/Gap Statement. Molecular mechanisms of azole resistance have been rarely reported for Trichosproron asahii. Similar to other fungi, we hypothesized that both ERG11 gene mutation and efflux pumps genes hyper-expression were implicated.
Aim. The current work aimed to study the sensitivity of clinical T. asahii isolates to different antifungal agents and to explore their resistance mechanisms by molecular methods including real-time PCR and gene sequencing.
Methods. The sensitivity of T. asahii isolates to fluconazole, amphotericin B and voriconazole was estimated by the Etest method. Real-time PCR was used to measure the relative expression of Pdr11, Mdr and ERG11 genes via the ACT1 housekeeping gene. Three pairs of primers were also chosen to sequence the ERG11 gene. This exploration was followed by statistical study including the receiver operating characteristic (ROC) curve analysis to identify a relationship between gene mean expression and the sensitivity of isolates.
Results. In 31 clinical isolates, the resistance frequencies were 87, 16.1 and 3.2 %, respectively, for amphotericin B, fluconazole and voriconazole. Quantitative real-time PCR demonstrated that only Mdr over-expression was significantly associated with FCZ resistance confirmed by univariate statistical study and the ROC curve analysis (P <0.05). The ERG11 sequencing revealed two mutations H380G and S381A in TN325U11 (MIC FCZ=8 µg ml−1) and H437R in TN114U09 (MIC FCZ=256 µg ml−1) in highly conserved regions (close to the haem-binding domain) but their involvement in the resistance mechanism has not yet been assigned.
Conclusion. T. asahii FCZ resistance mechanisms are proven to be much more complex and gene alteration sequence and/or expression can be involved. Only Mdr gene over-expression was significantly associated with FCZ resistance and no good correlation was observed between FCZ and VCZ MIC values and relative gene expression. ERG11 sequence alteration seems to play a major role in T. asahii FCZ resistance mechanism but their involvement needs further confirmation.
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Ex vivo potential of a quinoline-derivative nail lacquer as a new alternative for dermatophytic onychomycosis treatment
Gabriella da Rosa Monte Machado, Bruna Pippi, Simone Berlitz, Denise Diedrich, Diego Defferrari, William Lopes, Simone Cristina Baggio Gnoatto, Irene Clemes Kulkamp-Guerreiro, Marilene Henning Vainstein, Mickael Jean, Pierre Van de Weghe, Saulo Fernandes de Andrade and Alexandre Meneghello FuentefriaIntroduction. Onychomycosis infections currently show a significant increase, affecting about 10 % of the world population. Trichophyton rubrum is the main agent responsible for about 80 % of the reported infections. The clinical cure for onychomycosis is extremely difficult and effective new antifungal therapy is needed.
Hypothesis/Gap Statement. Ex vivo onychomycosis models using porcine hooves can be an excellent alternative for evaluating the efficacy of new anti-dermatophytic agents in a nail lacquer.
Aim. Evaluation of the effectiveness of a nail lacquer containing a quinoline derivative on an ex vivo onychomycosis model using porcine hooves, as well as the proposal of a plausible antifungal mechanism of this derivative against dermatophytic strains.
Methodology. The action mechanism of a quinoline derivative was evaluated through the sorbitol protection assay, exogenous ergosterol binding, and the determination of the dose-response curves by time-kill assay. Scanning electron microscopy evaluated the effect of the derivative in the fungal cells. The efficacy of a quinoline-derivative nail lacquer on an ex vivo onychomycosis model using porcine hooves was evaluated as well.
Results. The quinoline derivative showed a time-dependent fungicidal effect, demonstrating reduction and damage in the morphology of dermatophytic hyphae. In addition, the ex vivo onychomycosis model was effective in the establishment of infection by T. rubrum.
Conclusion. Treatment with the quinoline-derivative lacquer showed a significant inhibitory effect on T. rubrum strain in this infection model. Finally, the compound presents high potential for application in a formulation such as nail lacquer as a possible treatment for dermatophytic onychomycosis.
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- Molecular and Microbial Epidemiology
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A large food-borne outbreak of campylobacteriosis in kindergartens and primary schools in Pescara, Italy, May–June 2018
Simona Sorgentone, Luca Busani, Paolo Calistri, Giorgio Robuffo, Stefania Bellino, Vicdalia Acciari, Maurizio Ferri, Caterina Graziani, Salvatore Antoci, Fabrizio Lodi, Valeria Alfonsi, Cesare Cammà, Paolo Fazii, Xanthi Andrianou, Francesca Cito, Giuliano Lombardi, Gabriella Centorotola, Massimo D’Amario, Nicola D’Alterio, Vincenzo Savini, Fabrizio De Massis, Anna Pelatti, Marco Di Domenico, Guido Di Donato, Elisabetta Di Giannatale, Lisa Di Marcantonio, Violeta Di Marzio, Gabriella Di Serafino, Anna Janowicz, Cristina Marfoglia, Francesca Marotta, Daniela Morelli, Giacomo Migliorati, Diana Neri, Francesco Pomilio, Silvia Scattolini, Giovanni Rezza, Antonio Caponetti, Patrizio Pezzotti and Giuliano GarofoloIntroduction. In May–June 2018, an outbreak of campylobacteriosis involved students and school staff from kindergartens and primary schools in Pescara, southern Italy.
Aim. We present details of the epidemiological and microbiological investigation, and the findings of the analytical study, as well as the implemented control measures.
Methodology. To identify possible risk factors associated with the observed outbreak, a case control study was conducted using a questionnaire to collect information on the date of symptoms onset, type and duration of symptoms, type of healthcare contact, school attendance, and food items consumed at school lunches during the presumed days of exposure. Attack rates were calculated for each date and school. Logistic regression models were used to estimate the odds ratios of being a case and the odds of illness by food items consumed, respectively. Moreover, we carried out a comparative genomic analysis using whole genome multilocus sequence typing (wgMLST) of Campylobacter jejuni strains isolated during the outbreak investigation to identify the source of the outbreak.
Results. Overall, 222 probable cases from 21 schools were identified, and C. jejuni was successfully isolated from 60 patients. The meals in the schools involved were provided by two cooking centres managed by a joint venture between two food companies. Environmental and food sampling, epidemiological and microbiological analyses, as well as a case control study with 176 cases and 62 controls from the same schools were performed to identify the source of the outbreak. The highest attack rate was recorded among those having lunch at school on 29 May (7.8 %), and the most likely exposure was ‘caciotta’ cheese (odds ratio 2.40, 95 % confidence interval 1.10–5.26, P=0.028). C. jejuni was isolated from the cheese, and wgMLST showed that the human and cheese isolates belonged to the same genomic cluster, confirming that the cheese was the vehicle of the infection.
Conclusion. It is plausible that a failure of the pasteurization process contributed to the contamination of the cheese batches. Timely suspension of the catering service and summer closure of the schools prevented further spread.
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Tuberculosis cases in a prison in the extreme south of Brazil
Introduction. Tuberculosis (TB) control is a challenge, especially in vulnerable populations, such as prisoners.
Hypothesis. In prison houses, the transmission of micro-organisms that cause infectious diseases can occur due to the susceptibility and immune compromise of prisoners, and due to the precarious physical conditions of the prison houses. However, strategies such as monitoring by health professionals, can mitigate the transmission of these micro-organisms, as well as, reduce the number of coinfections and antimicrobials resistance.
Aim. This study attempted to analyse the dynamics of transmission and the antimicrobial resistance profile of Mycobacterium tuberculosis strains obtained from prisoners and to characterize the epidemiological, clinical and laboratory profiles of prisoners diagnosed with TB.
Methodology. A cross-sectional and retrospective study was conducted with sputum samples collected from 228 distinct prisoners who were treated at the Health Unit located in the Regional Penitentiary of Rio Grande, Rio Grande do Sul, Brazil. The antimicrobial resistance profile of the strains was evaluated using the Resazurin Microtiter Assay and the transmission dynamics was investigated using 15-loci MIRU-VNTR.
Results. Thirty-five patients (15.4 %) were diagnosed with TB, and when a TB/HIV coinfection was assessed, 8.6 % (3/35) of the patients were positive. In addition, all patients with results available for HBV, HCV, syphilis and diabetes mellitus were negative. Based on the genotypic profile, 55.9 % of the clinical isolates were grouped into five groups. One isolate with mono-resistance to isoniazid and two with mono-resistance to streptomycin were found.
Conclusion. The presence of a Health Unit may have influenced the low numbers of TB/HIV, TB/HBV, TB/HCV, TB/syphilis coinfections and TB cases resistant to antimicrobials. Recent M. tuberculosis transmission can be inferred based on the high percentage of formatting of clusters. This situation stresses the need to improve active and passive detection, the screening of individuals for TB upon entrance into prison for early detection, and the implementation of prophylactic measures to reduce M. tuberculosis transmission.
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Molecular characterization of carbapenem-resistant Enterobacteriaceae and emergence of tigecycline non-susceptible strains in the Henan province in China: a multicentrer study
Introduction. Carbapenem-resistant Enterobacteriaceae (CRE) have been responsible for nosocomial outbreaks worldwide and have become endemic in several countries.
Hypothesis/Gap Statement. To better understand the epidemiological trends and characteristics of CRE in the Henan province.
Aim. We assessed the molecular epidemiological characteristics of 305 CRE strains isolated from patients in 19 secondary or tertiary hospitals in ten areas of the Henan province in China.
Methodology. A total of 305 CRE isolates were subjected to multiple tests, including in vitro antimicrobial susceptibility testing, PCR for carbapenemase genes bla KPC, bla NDM, bla IMP, bla VIM, bla OXA-48-like. Tigecycline-resistant genes ramR, oqxR, acrR, tetA, rpsJ, tetX, tetM, tetL were analysed in five tigecycline non-susceptible carbapenem-resistant Klebsiella pneumoniae isolates (TNSCRKP). Additionally, multilocus sequence typing (MLST) was performed for carbapenem-resistant K. pneumoniae (CRKP).
Results. The most common CRE species were K. pneumoniae (234, 77 %), Escherichia coli (36, 12 %) and Enterobacter cloacae (13, 4 %). All strains exhibited multi-drug resistance. Overall, 97 % (295/305) and 97 % (297/305) of the isolates were susceptible to polymyxin B and tigecycline, respectively. A total of 89 % (271/305) of the CRE isolates were carbapenemase gene-positive, including 70 % bla KPC, 13 % bla NDM, 6 % bla IMP, and 1 % combined bla KPC/bla NDM genes. K. pneumoniae carbapenemase (KPC) was the predominant carbapenemase in K. pneumoniae (87 %), whereas NDM and IMP were frequent in E. coli (53 %) and E. cloacae (69 %), respectively. Mutations in the ramR, tetA, and rpsJ genes were detected in five TNSCRKP. Moreover, 15 unique sequence types were detected, with ST11 (74 %), ST15 (9 %) and ST2237 (5 %) being dominant among K. pneumoniae strains.
Conclusion. A high proportion of CRE strains were carbapenemase-positive, and five carbapenem-resistant K. pneumonia isolates were tigecycline non-susceptible, indicating a need for the ongoing surveillance of CRE and effective measures for the prevention of CRE infections.
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Adaptations of carbapenem resistant Acinetobacter baumannii (CRAB) in the hospital environment causing sustained outbreak
More LessIntroduction. Carbapenem resistance in Acinetobacter baumannii ( A. baumannii ) is an emerging global threat.
Gap statement. The adaptation strategies of A. baumannii for this emergence as a nosocomial pathogen has been less studied.
Aim. This prospective study analysed a sustained outbreak of carbapenem resistant Acinetobacter baumannii (CRAB) in the intensive care unit (ICU) with reference to antimicrobial resistance and virulence in the colonizing and pathogenic isolates under carbapenem stress.
Results. The CRAB isolates from initial and sustained outbreak were found harbouring multiple carbapenemase genes. These genes included bla OXA-23 ,bla IMP, bla VIM and bla NDM. From NICU environment three phenotypically carbapenem susceptible isolates were found carrying bla OXA-23, bla IMP, bla VIM genes. Prior imipenem therapy was one of the risk factors (P=0.0016). The outbreak was polyclonal. Under imipenem stress, outbreak isolates showed no loss of carbapenemase genes against stress free conditions (23.7±1.33 days). Biofilm formation increased with imipenem concentration, with outbreak isolates producing highest biomass. While the pathogens showed a slow growth rate on imipenem exposure, the colonisers grew rapidly (P <0.0001).
Methods. Sustained outbreak of CRAB was identified in the ICU (July 2015 to December 2017). Risk factors for acquisition of CRAB was studied. A. baumannii isolates were also collected from the environments of ICU and neonatal ICU (NICU) and blood cultures of septic neonates. Isolates were characterized based on antimicrobial susceptibility, genetic profile, integrons carriage and clonality. Biofilm formation and growth kinetics were studied under varying carbapenem stress.
Conclusion. Intense carbapenem exposure in the ICU facilitates persistence of CRAB by several adaptations causing sustained outbreaks.
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- Pathogenesis, Virulence and Host Response
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Host factors abolish the need for polysaccharides and extracellular matrix-binding protein in Staphylococcus epidermidis biofilm formation
Introduction. Staphylococcus epidermidis is predominant in implant-associated infections due to its capability to form biofilms. It can deploy several strategies for biofilm development using either polysaccharide intercellular adhesin (PIA), extracellular DNA (eDNA) and/or proteins, such as the extracellular matrix-binding protein (Embp).
Hypothesis/Gap Statement. We hypothesize that the dichotomic regulation of S. epidermidis adhesins is linked to whether it is inside a host or not, and that in vitro biofilm investigations in laboratory media may not reflect actual biofilms in vivo.
Aim. We address the importance of PIA and Embp in biofilm grown in ‘humanized’ media to understand if these components play different roles in biofilm formation under conditions where bacteria can incorporate host proteins in the biofilm matrix.
Methodology. S. epidermidis 1585 WT (deficient in icaADBC), and derivative strains that either lack embp, express embp from an inducible promotor, or express icaADBC from a plasmid, were cultivated in standard laboratory media, or in media with human plasma or serum. The amount, structure, elasticity and antimicrobial penetration of biofilms was quantified to describe structural differences caused by the different matrix components and growth conditions. Finally, we quantified the initiation of biofilms as suspended aggregates in response to host factors to determine how quickly the cells aggregate in response to the host environment and reach a size that protects them from phagocytosis.
Results. S. epidermidis 1585 required polysaccharides to form biofilm in laboratory media. However, these observations were not representative of the biofilm phenotype in the presence of human plasma. If human plasma were present, polysaccharides and Embp were redundant for biofilm formation. Biofilms formed in human plasma were loosely attached and existed mostly as suspended aggregates. Aggregation occurred after 2 h of exposing cells to plasma or serum. Despite stark differences in the amount and composition of biofilms formed by polysaccharide-producing and Embp-producing strains in different media, there were no differences in vancomycin penetration or susceptibility.
Conclusion. We suggest that the assumed importance of polysaccharides for biofilm formation is an artefact from studying biofilms in laboratory media void of human matrix components. The cell–cell aggregation of S. epidermidis can be activated by host factors without relying on either of the major adhesins, PIA and Embp, indicating a need to revisit the basic question of how S. epidermidis deploys self-produced and host-derived matrix components to form antibiotic-tolerant biofilms in vivo.
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Loss of the virulence plasmid by Shigella sonnei promotes its interactions with CD207 and CD209 receptors
Introduction. Shigella sonnei, the cause of bacillary dysentery, belongs to Gram-negative enteropathogenic bacteria. S. sonnei contains a 210 kb virulence plasmid that encodes an O-antigen gene cluster of LPSs. However, this virulence plasmid is frequently lost during replication. It is well-documented that after losing the O-antigen and becoming rough strains, the Gram-negative bacteria may express an LPS core on its surface. Previous studies have suggested that by using the LPS core, Gram-negative bacteria can interact with several C-type lectin receptors that are expressed on antigen-presenting cells (APCs).
Hypothesis/Gap Statement. S. sonnei by losing the virulence plasmid may hijack APCs via the interactions of LPS-CD209/CD207.
Aim. This study aimed to investigate if the S. sonnei rough strain, by losing the virulence plasmid, interacted with APCs that express C-type lectins of human CD207, human CD209a and mouse CD209b.
Methodology. SDS-PAGE silver staining was used to examine the O-antigen expression of S. sonnei WT and its rough strain. Invasion assays and inhibition assays were used to examine the ability of S. sonnei WT and its rough strain to invade APCs and investigate whether CD209 and CD207 are receptors for phagocytosis of rough S. sonnei . Animal assays were used to observe the dissemination of S. sonnei .
Results. S. sonnei did not express O-antigens after losing the virulence plasmid. The S. sonnei rough strain invades with APCs, including human dendritic cells (DCs) and mouse macrophages. CD209 and CD207 are receptors for phagocytosis of rough S. sonnei . Expression of the O-antigen reduces the ability of the S. sonnei rough strain to be disseminated to mesenteric lymph nodes and spleens.
Conclusion. This work demonstrated that S. sonnei rough strains – by losing the virulence plasmid – invaded APCs through interactions with CD209 and CD207 receptors.
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Citrobacter koseri stimulates dendritic cells to induce IL-33 expression via abundant ATP production
More LessIntroduction. Food allergies (FAs) occur due to intestinal immune dysfunction elicited by dysbiotic conditions. It was previously determined by us that Citrobacter species propagate in the faeces of mice with FAs and worsen allergic symptoms by inducing the allergenic cytokine IL-33. Dendritic cells can play important roles in regulation of FA responses.
Hypothesis. Citrobacter species propagating in intestines of mice worsen allergic symptoms by stimulating dendritic cells to induce IL-33 expression.
Aim. The aim of the present study was to analyse whether C. koseri stimulates dendritic cells to induce IL-33 expression.
Methodology. IL-33 expression was evaluated in a DC2.4 mouse dendritic cell line stimulated by live or heat-inactivated C. koseri JCM1658, ATP, LPS extracted from C. koseri JCM1658 or other enterobacteria by real-time PCR. The ATP concentration and number of live bacteria in the culture supernatant were measured simultaneously.
Results. Live C. koseri JCM1658 induced higher levels of IL-33 expression than other enterobacteria tested, but such a response was not elicited by heat-inactivated C. koseri JCM1658. LPS extracted from C. koseri JCM1658 did not induce IL-33 expression and suppressed live C. koseri JCM1658-induced IL-33 expression via the activation of Toll-like receptor 4 signalling. Furthermore, ATP produced by C. koseri JCM1658 stimulated dendritic cells to induce IL-33 expression by stimulating the P2X7 receptor, and LPS attenuated extracellular ATP-induced IL-33 expression. C. koseri JCM1658 was observed to proliferate more vigorously and produce more ATP than other enterobacteria.
Conclusion. C. koseri acts as an allergenic bacterium through ATP production, stimulating dendritic cells to induce IL-33 expression, while LPS released from inactivated C. koseri JCM1658 attenuates this allergenicity.
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Immunomodulatory streptococci that inhibit CXCL8 secretion and NFκB activation are common members of the oral microbiota
Introduction. Oral tissues are generally homeostatic despite exposure to many potential inflammatory agents including the resident microbiota. This requires the balancing of inflammation by regulatory mechanisms and/or anti-inflammatory commensal bacteria. Thus, the levels of anti-inflammatory commensal bacteria in resident populations may be critical in maintaining this homeostatic balance.
Hypothesis/Gap Statement. The incidence of immunosuppressive streptococci in the oral cavity is not well established. Determining the proportion of these organisms and the mechanisms involved may help to understand host-microbe homeostasis and inform development of probiotics or prebiotics in the maintenance of oral health.
Aim. To determine the incidence and potential modes of action of immunosuppressive capacity in resident oral streptococci.
Methodology. Supragingival plaque was collected from five healthy participants and supragingival and subgingival plaque from five with gingivitis. Twenty streptococci from each sample were co-cultured with epithelial cells±flagellin or LL-37. CXCL8 secretion was detected by ELISA, induction of cytotoxicity in human epithelial cells by lactate dehydrogenase release and NFκB-activation using a reporter cell line. Bacterial identification was achieved through partial 16S rRNA gene sequencing and next-generation sequencing.
Results. CXCL8 secretion was inhibited by 94/300 isolates. Immunosuppressive isolates were detected in supragingival plaque from healthy (4/5) and gingivitis (4/5) samples, and in 2/5 subgingival (gingivitis) plaque samples. Most were Streptococcus mitis/oralis. Seventeen representative immunosuppressive isolates all inhibited NFκB activation. The immunosuppressive mechanism was strain specific, often mediated by ultra-violet light-labile factors, whilst bacterial viability was essential in certain species.
Conclusion. Many streptococci isolated from plaque suppressed epithelial cell CXCL8 secretion, via inhibition of NFκB. This phenomenon may play an important role in oral host-microbe homeostasis.
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Normal breathing releases SARS-CoV-2 into the air
Piero Di Carlo, Katia Falasca, Claudio Ucciferri, Bruna Sinjari, Eleonora Aruffo, Ivana Antonucci, Alessandra Di Serafino, Arianna Pompilio, Verena Damiani, Domitilla Mandatori, Simone De Fabritiis, Beatrice Dufrusine, Emily Capone, Piero Chiacchiaretta, William H. Brune, Giovanni Di Bonaventura and Jacopo VecchietThis study tests the release of SARS-CoV-2 RNA into the air during normal breathing, without any sign of possible risk of contagion such as coughing, sneezing or talking. Five patients underwent oropharyngeal, nasopharyngeal and salivary swabs for real-time reverse transcriptase PCR (RT-PCR) detection of SARS-CoV-2 RNA. Direct SARS-CoV-2 release during normal breathing was also investigated by RT-PCR in air samples collected using a microbiological sampler. Viral RNA was detected in air at 1 cm from the mouth of patients whose oropharyngeal, nasopharyngeal and salivary swabs tested positive for SARS-CoV-2 RNA. In contrast, the viral RNA was not identified in the exhaled air from patients with oropharyngeal, nasopharyngeal and salivary swabs that tested negative. Contagion of SARS-CoV-2 is possible by being very close to the mouth of someone who is infected, asymptomatic and simply breathing.
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Profiles of mutations in hepatitis B virus surface and polymerase genes isolated from treatment-naïve Nigerians infected with genotype E
Introduction. Hepatitis B virus (HBV) infection is the leading cause of hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). HBV genotype E (HBV/E) is the predominant genotype in West Africa and has been linked epidemiologically with chronic and occult HBV infections as well as development of HCC. Mutations in the surface and polymerase genes of HBV have been associated with occult infection, drug resistance, vaccine escape, as well as HCC.
Hypothesis/Gap Statement. There is limited data on the occurrence and patterns of mutations associated with occult infection, drug resistance, vaccine escape and HCC for HBV/E.
Aim. This study characterized amino acid (aa) substitutions in the major hydrophilic (MHR) and reverse transcriptase (RT) regions of the surface and polymerase genes respectively of HBV sequences from a group of Nigerians with genotype E infection. The CpG islands of the PreC/C and PreS/S regions of these sequences were also described.
Methodology. HBV surface and polymerase genes were detected using PCR techniques. Occurrence of new and previously described mutations in these genes were analysed using phylogenetic techniques.
Results. Overall 13 HBV isolates were each sequenced for polymerase and surface genes mutations. Thirteen and nine PreS/S and PreC/C HBV genes respectively were analysed for CpG islands. Mutations in the MHR and a-determinants region of the S protein were discovered in eleven and nine of the 13 tested isolates respectively. These mutations were concomitant with aa changes in the RT functional domains of the isolates. Mutations associated with vaccine escape, occult infection and poor HCC prognosis were identified in HBV/E isolated in this study. Furthermore, all the isolates had at least one putative nucleotide analogue resistance mutations. Drug resistance mutations had the highest association with CpG islands.
Conclusion. The results of this study contribute to further understanding of HBV variability in Nigeria and the West African region. This will aid the planning of adequate HBV immunization and treatment programmes for the countries in the region.
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- Prevention, Therapy and Therapeutics
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In vitro synergy of 5-nitrofurans, vancomycin and sodium deoxycholate against Gram-negative pathogens
More LessIntroduction. There is an urgent need for effective therapies against bacterial infections, especially those caused by antibiotic-resistant Gram-negative pathogens.
Hypothesis. Synergistic combinations of existing antimicrobials show promise due to their enhanced efficacies and reduced dosages which can mitigate adverse effects, and therefore can be used as potential antibacterial therapy.
Aim. In this study, we sought to characterize the in vitro interaction of 5-nitrofurans, vancomycin and sodium deoxycholate (NVD) against pathogenic bacteria.
Methodology. The synergy of the NVD combination was investigated in terms of growth inhibition and bacterial killing using checkerboard and time-kill assays, respectively.
Results. Using a three-dimensional checkerboard assay, we showed that 5-nitrofurans, sodium deoxycholate and vancomycin interact synergistically in the growth inhibition of 15 out of 20 Gram-negative strains tested, including clinically significant pathogens such as carbapenemase-producing Escherichia coli , Klebsiella pneumoniae and Acinetobacter baumannii , and interact indifferently against the Gram-positive strains tested. The time-kill assay further confirmed that the triple combination was bactericidal in a synergistic manner.
Conclusion. This study demonstrates the synergistic effect of 5-nitrofurans, sodium deoxycholate and vancomycin against Gram-negative pathogens and highlights the potential of the combination as a treatment for Gram-negative and Gram-positive infections.
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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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