- Volume 68, Issue 3, 2019
Volume 68, Issue 3, 2019
- Editorial
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- Letters
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Xpert MRSA screening in surgical patient flow; time for a rethink for hub-and-spoke laboratory models?
More LessThe move towards pathology networks and hub-and-spoke models of medical laboratory service provision has significantly changed the flow of samples, and the impact of results on patients, over recent years. At the same time advances in technology, including rapid, simple to use molecular platforms, are changing the way microbiology results can be utilized. Like many other medical microbiology laboratories, we struggle with this balance for many different sample types and test requests. Work published by Neilson et al. in Journal of Medical Microbiology last year looked at this balance for methicillin-resistant Staphylococcus aureus (MRSA) genotypic diagnostics and suggested significant cost savings when a whole-healthcare economy perspective was adopted. However, as with all changes, implementing MRSA molecular diagnostics in different clinical settings must be considered carefully. We add to this discussion in our accompanying letter, detailing our experience (in a hub-and-spoke medical microbiology laboratory setting) of ‘rapid’ MRSA molecular diagnostics for day-case surgery where pre-operative assessment had been missed, exploring the impact and costs of these tests. We find no impact on patient care, but at considerable additional cost. We hope this will add a cautionary note to those considering implementing molecular microbiology diagnostics, and reopen the debate on where, in hub-and-spoke laboratory models, such devices should be situated.
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- Review
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The rise of pneumonic plague in Madagascar: current plague outbreak breaks usual seasonal mould
Madagascar has just emerged from the grip of an acute urban pneumonic plague outbreak, which began in August 2017, before the usual plague season of October–April and outside the traditional plague foci in the northern and central highlands. The World Health Organization reported a total of 2417 confirmed, probable and suspected cases, including 209 deaths between 1 August and 26 November 2017. The severity and scope of this outbreak, which has affected those in higher socioeconomic groups as well as those living in poverty, along with factors including the potential for use of multi-drug-resistant strains of plague in bioterrorism, highlights the ongoing threat posed by this ancient disease. Factors likely to have contributed to transmission include human behaviour, including burial practices and movement of people, poor urban planning leading to overcrowding and ready transmission by airborne droplets, climatic factors and genomic subtypes. The outbreak demonstrates the importance of identifying targeted pneumonic plague therapies and of developing vaccines that can be administered in planned programmes in developing countries such as Madagascar where plague is endemic. The dominance of pneumonic plague in this outbreak suggests that we need to focus more urgently on the danger of person-to-person transmission, as well as the problem of transmission of plague from zoonotic sources.
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- Antimicrobial Resistance
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Utilizing genomic analyses to investigate the first outbreak of van A vancomycin-resistant Enterococcus in Australia with emergence of daptomycin non-susceptibility
Introduction. The majority of vancomycin-resistant Enterococcus faecium (VREfm) in Australia is of the vanB genotype. An outbreak of vanA VREfm emerged in our haematology/oncology unit between November 2014 and May 2015. The first case of daptomycin non-susceptible E. faecium (DNSEfm) detected was a patient with vanA VREfm bacteraemia who showed clinical failure of daptomycin therapy, prompting microbiologic testing confirming daptomycin non-susceptibility.
Objectives. To describe the patient profiles, antibiotic susceptibility and genetic relatedness of vanA VREfm isolates in the outbreak.
Methods. Chart review of vanA VREfm colonized and infected patients was undertaken to describe the demographics, clinical features and outcomes of therapy. Whole genome sequencing of vanA VREfm isolates involved in the outbreak was conducted to assess clonality.
Results. In total, 29 samples from 24 patients tested positive for vanA VREfm (21 screening swabs and 8 clinical isolates). Five isolates were DNSEfm (four patients colonized, one patient with bacteraemia), with only one patient exposed to daptomycin previously. In silico multi-locus sequence typing of the isolates identified 25/26 as ST203, and 1/26 as ST796. Comparative genomic analysis revealed limited core genome diversity amongst the ST203 isolates, consistent with an outbreak of a single clone of vanA VREfm.
Conclusions. Here we describe an outbreak of vanA VREfm in a haematology/oncology unit. Genomic analysis supports transmission of an ST203 vanA VRE clone within this unit. Daptomycin non-susceptibility in 5/24 patients left linezolid as the only treatment option. Daptomycin susceptibility cannot be assumed in vanA VREfm isolates and confirmatory testing is recommended.
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Synergistic activity of polymyxin B combined with vancomycin against carbapenem-resistant and polymyxin-resistant Acinetobacter baumannii: first in vitro study
Purpose. The effect of a combination of polymyxin B (PMB) and vancomycin (VAN) was assessed against six Acinetobacter baumannii clinical isolates belonging to six different clusters (three PMB-susceptible and three PMB-resistant).
Methodology. The synergistic effect of the PMB–VAN combination was determined with the checkerboard, time-kill, disk-diffusion and M.I.C.Evaluator assays. PMB-resistance was investigated with mcr-1 gene amplification and a mutant frequency assay.
Results. In the checkerboard assay, all PMB-resistant isolates showed a synergistic effect. The time-kill assay demonstrated that the PMB–VAN combination had a bactericidal effect at 24 h against isolates with a high mutant rate for PMB, suggesting that this combination may block the hypermutation of some isolates. No antagonism was detected. All PMB-resistant isolates also showed synergism in the disk-diffusion test, and a significant decrease in VAN MICs in the M.I.C.Evaluator assay.
Conclusion. Our findings indicate that the PMB–VAN combination has a synergistic effect on A. baumannii , especially against PMB-resistant isolates.
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Genotypic and phenotypic variations in methicillin-resistant Staphylococcus aureus isolates from outpatient, inpatient and nursing homes
More LessPurpose. The epidemiological shift in MRSA distribution from healthcare-related facilities to the general population is distressing and requires continuous monitoring to manage and control the rate of incidences.
Method. The retrospective relationship between genetic and phenotypic variability of methicillin-resistant Staphylococcus aureus (MRSA) isolates was determined in respect to the specimen source, patient location, sex and age. A total of 521 MRSA isolates were classified based on SCCmec, mec, agr, pvl and spa genetic markers using three different multiplex PCRs.
Results. Based on the genetic variability, the isolates were divided into 97 profiles, of which 59% belonged to only two profiles (P17 and P33). P17 was the predominate profile, harbouring SCCmecIVa, ccr2, mecB, agr1, spa413 and pvl markers. P17 was more prevalent among the younger population (average 33.9 years) from outpatient (77%) locations and wound (88%) sources. The second largest profile was P33, harbouring SCCmecII, ccr2+ccr3, mecA, agr2, spa413 and no PVL. P33 was more prevalent in the older population (average 70.7 years) and more common in females (62%) than males (38%). With respect to antibiotic resistance, P33 exhibited a high rate of resistance to penicillins, cephalosporins, fluoroquinolones and macrolides, and P17 had a lower resistance to fluoroquinolones.
Conclusion. This report contributes to the existing understanding of evolutionary epidemiology of antibiotic resistance in MRSA. The diversity of MRSA isolates and unique environmental preferences for each profile highlights the importance of epidemiological knowledge of MRSA distribution to determine the best treatment for patients in both community and hospital settings.
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The clinical significance of carbapenem-resistant Klebsiella pneumoniae rectal colonization in critically ill patients: from colonization to bloodstream infection
Purpose . To highlight the clinical significance of carbapenem-resistant Klebsiella pneumoniae (CRKP) rectal colonization by examining the risk factors for CRKP rectal colonization and subsequent bloodstream infection (BSI) in critically ill patients.
Methodology . Prospective study of CRKP rectal colonization in an intensive care unit (ICU) during a 39-month period. CRKP strains isolated from both the blood cultures and corresponding rectal specimens (n=96) of patients were screened by PCR for the presence of antibiotic resistance-associated genes. Molecular analyses were conducted to investigate the clonal relatedness of CRKP strains from the rectal and blood specimens.
Results . Among the 498 patients, 226 were rectally colonized by CRKP, 48 of whom developed a CRKP BSI. The median time from hospital admission to the detection of CRKP rectal colonization was 8 days, while the median time from colonization to BSI was 4 days. The duration of ICU stay, patient/nurse ratio and prior use of antianaerobic antimicrobials were associated with CRKP rectal colonization. No specific factor was associated with BSIs in the colonized patients. The bla KPC-2 gene was detected in all 96 strains, which were all classified as sequence type ST-258. Representative pairs (n=48) of CRKP strains colonizing and infecting the same patient shared the same pulsotype.
Conclusion . Our results indicate that hospitalized patients become infected with their colonizing strains, supporting the strong association between colonization and BSI. Limiting antianaerobic antimicrobial administration, reducing the duration of ICU stay and maintaining a low patient/nurse ratio are possible strategies to restrict rectal CRKP colonization in ICUs.
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Acquired qnrVC1 and bla NDM-1 resistance markers in an international high-risk Pseudomonas aeruginosa ST773 clone
More LessA multidrug-resistant Pseudomonas aeruginosa PS1 isolated from urine clinical sample was investigated in this study. The strain exhibited resistance to piperacillin/tazobactam, ciprofloxacin, imipenem, ceftazidime but it was susceptible to colistin. Analysis of whole-genome sequencing data by ResFinder detected various resistance determinants including qnrVC1 and bla NDM-1. The multiresistant P. aeruginosa isolate belonged to ST773 high-risk clone. The qnrVC1 and bla NDM-1 determinants were incorporated into different integrons. Expression of bla NDM-1 was fourfold and qnrVC1 was twofold increased, compared to that of rpsL housekeeping gene. Mutations in gyrA Thr83Leu and parC Ser87Leu were detected and additionally qnrVC1 expression indicates protective effect of QnrVC1 pentapeptid protein on gyrase and topoisomerase. High-risk P. aeruginosa clones integrate various carbapenemase and other resistance determinants into their genomes that facilitates further dissemination of multiresistance among clinical isolates. We report bla NDM-1 and qnrVC1 genes in P. aeruginosa ST773 international high-risk clone.
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Prevalence and genetic characterization of cephalosporin-resistant Enterobacteriaceae among dogs and cats in an animal shelter
More LessPurpose . The prevalence of antimicrobial-resistant bacteria, especially cephalosporin-resistant Enterobacteriaceae , is a major concern for human and animal health. We investigated the prevalence of cephalosporin-resistant Enterobacteriaceae among sheltered dogs and cats with various backgrounds.
Method . Faecal samples or rectal swabs were collected from 151 dogs and 182 cats, and screened for the presence of antimicrobial-resistant bacteria. Isolates were characterized phenotypically and genotypically by pulsed-field gel electrophoresis, multi-locus sequence typing and phylogenetic grouping. The animal attributes related to bacterial carriage were statistically analysed.
Results . Cephalosporin-resistant Enterobacteriaceae was detected in 22 dogs (14.6%) and 20 cats (11.0%): 21 were extended-spectrum β-lactamase (ESBL)-producing, 20 were AmpC-producing, and 1 was both ESBL- and AmpC-producing. Their β-lactamase genes were varied and associated with humans, animals or other origins. The genes CTX-M-14 (n=9) and CMY-2 (n=9) were dominant, but CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-15, CTX-M-24, CTX-M-27, CTX-M-55 and DHA-1 genes were also detected. Genotyping of isolates revealed that β-lactamase-producing Enterobacteriaceae had high genetic diversity. Relationships between animals harbouring cephalosporin-resistant Enterobacteriaceae and individual attributes, such as sex and nutrition type, were detected, but there was no correlation between history of human association and the presence of the bacterium in either dogs or cats.
Conclusion . We found several types of cephalosporin-resistant Enterobacteriaceae distributed among companion animals with a range of individual attributes and histories in Osaka, Japan. Companion animals may play a bridging role in the circulation of antimicrobial-resistant bacteria from humans and from other origins.
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Antifungal susceptibilities, biofilms, phospholipase and proteinase activities in the Candida rugosa complex and Candida pararugosa isolated from tertiary teaching hospitals
Purpose. Non-albicans Candida species have emerged as fungal pathogens that cause invasive infections, with many of these species displaying resistance to commonly used antifungal agents. This study was confined to studying the characteristics of clinical isolates of the C. rugosa complex and C. pararugosa species.
Methodology. Seven isolates of the C. rugosa complex and one isolate of C. pararugosa were obtained from two tertiary referral hospitals in Malaysia. Their antifungal susceptibilities, biofilm, proteinase, phospholipase, esterase and haemolysin activities were characterized. Biofilms were quantified using crystal violet (CV) and tetrazolium (XTT) reduction assays at 1.5, 6, 18, 24, 48 and 72 h.
Results/Key findings. The E-test antifungal tests showed that both species have elevated MICs compared to C. albicans and C. tropicalis. The highest biomass was observed in one of the C. rugosa isolates (0.237), followed by C. pararugosa (0.206) at 18 h of incubation. However, the highest bioactivity was observed in the C. rugosa ATCC 10571 strain at 24 h (0.075), followed by C. pararugosa at 48 h (0.048) and the same C. rugosa strain at 24 h (0.046), with P<0.05. All isolates exhibited high proteinase activity (+++) whereas six isolates showed very strong esterase activity (++++). All the isolates were alpha haemolytic producers. None of the isolates exhibited phospholipase activity.
Conclusion. Elevated MICs were shown for the C. rugosa complex and C. pararugosa for commonly used antifungal drugs. Further studies to identify virulence genes involved in the pathogenesis and genes that confer reduced drug susceptibility in these species are proposed.
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- Clinical Microbiology
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Characterization of the mechanism and impact of staphylokinase on the formation of Candida albicans and Staphylococcus aureus polymicrobial biofilms
More LessPurpose. Candida albicans and Staphylococcus aureus can be co-isolated in biofilm-associated infections. However, treatments have not been well established due to a lack of antibiofilm strategies. Hence, this study aims to characterize the mechanism and impact of Staphylokinase (Sak) on fungal-bacterial polymicrobial biofilms.
Methodology. Sak generation levels were obtained via chromogenic analysis. C. albicans and S. aureus polymicrobial biofilm formation and integrity were analysed using a bright-field microscope and scanning electron microscopy (SEM). Metabolic mitochondrial activity, growth rate and adhesive capacity were also measured. Quantification real-time RT-PCR (qRT-PCR) was carried out to evaluate the expression levels of biofilm-related genes. Furthermore, the biofilm inhibitory potential of Sak alone or combined with antimicrobials was investigated.
Results. Sak production levels varied, ranging from 0.130 to 0.648. A strong decrease of biomass, metabolic activity andearly stage growth rate was demonstrated in the Sak-treated group. SEM showed S. aureus attached on hyphae of C. albicans in sporadic small microcolonies after Sak treatment. Moreover, the gene expression levels of HWP1, EFG1 and NRG1 were significantly altered, while no obvious difference was observed in ALS3. Finally, Sak had a notable impact on mature polymicrobial biofilms alone or when combined with vancomycin and fluconazole.
Conclusion.The effect induced by Sak to C. albicans and S. aureus polymicrobial biofilms is caused by decreased biomass, biofilm integrity, metabolic activity and early stage growth rate. Alterations of gene expression levels were consistent with Sak-induced phenotypic change. Combined treatment strategies are essential for optimal activities against fungal-bacterial polymicrobial biofilms.
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Detection and identification of bacterial pathogens directly from sputum samples by pyrosequencing
More LessPurpose. The standard culture findings for detecting and identifying bacterial pathogens in patients with lower respiratory tract infections (LRTIs) are usually not available for two to three days, which delays the initiation of appropriate antibiotic therapies. We aimed to develop a faster method of identification of bacterial pathogens in LRTIs which would offer a timelier guide to initial antibiotic choices.
Methodology. The developed PCR-pyrosequencing-based method was defined as mask PCR-pyrosequencing (MPP). This method uses primer pairs deliberately designed to mask the interference of colonised bacteria in sputum to detect and identify bacterial pathogens directly from LRTI patient sputum samples within 5 h. Accordingly, the standard PCR-pyrosequencing method was defined as normal PCR-pyrosequencing (NPP) here. The clinical performance of the novel system was evaluated by comparing with traditional semi-quantitative culture and identification results.
Results. The coincidence for culture and MPP was 91.3 %. Compared with the semi-quantitative culture results, NPP identified 89.9 % strains of grade 3+ (corresponding to 1.0×106 c.f.u ml−1) and 100 % of grade 4+ (corresponding to 1.0×107 c.f.u ml−1), both of which were considered to be the presumptive pathogens in the clinics. MPP identified 98.9 % strains of grade 3+ and 100 % of grade 4+. Additionally, PCR-pyrosequencing could detect a minimum concentration of 1.0×106 c.f.u ml−1 of bacteria in sputum, with no significant difference between NPP and MPP.
Conclusion. The PCR-pyrosequencing technique developed in this study is an accurate, fast, and high throughput method for the direct detection and identification of bacterial pathogens from sputum.
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Molecular characterization and genotyping of methicillin-resistant Staphylococcus aureus in nasal carriage of healthy Iranian children
More LessPurpose . Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a considerable public health concern in both developed and developing countries due to the rapid spread of this bacterium around the world, also the epidemiology of MRSA has changed, as the isolation of MRSA strains is not limited to health-care settings or patients with predisposing risk factors. Therefore, the objective of this study is to determine the genetic diversity and antibiotic resistance profile of CA-MRSA nasal carriage in Iranian children.
Methodology . A cross-sectional study was conducted from April 2013 to March 2014. A total of 25 CA-MRSA were isolated from the anterior nares of 410 preschool children with no risk factors. All MRSA isolates were characterized by detection of the Panton–Valentine leukocidin (pvl) and γ-hemolysin genes, staphylococcal cassette chromosome mec (SCCmec) typing and multi-locus sequence typing (MLST).
Results . In 25 CA-MRSA isolates, Pvl and γ-hemolysin genes were detected in one (4%) and 18 (72 %) isolates; respectively. Overall, 92% (23/25) of isolates belonged to SCCmec type IV and 8% (2/25) of them had SCCmec type V profile. Using MLST, the 25 isolates were grouped into six clonal complexes (CC) and eight sequence types (ST) (CC5/ST6, CC22/ST22 and ST217, CC30/ST30 and ST1107, CC78/ST859, CC398/ST291 and CC97/ST405). The ST859/SCCmec IV (11/25, 44%) was the predominant clone among the isolates. ST859-MRSA-IV-pvl-negative (resistant to tetracycline) have successfully adapted to the Iranian preschool children population.
Conclusion . Our results suggest that the genomic diversity was observed among the CA-MRSA. In addition, the current study demonstrates that pvl is not a reliable marker for CA-MRSA in our region.
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Evaluation of a novel immunochromatographic lateral flow assay for rapid detection of OXA-48, NDM, KPC and VIM carbapenemases in multidrug-resistant Enterobacteriaceae
Purpose. The global spread and increasing clinical impact of carbapenemase-producing bacteria are alarming. Rapid diagnostic techniques for carbapenemase detection are of the utmost importance to prevent delays in efficient antibiotic therapy and the control of spread in hospitals. Recently, multiplex immunochromatographic lateral flow tests (ICTs) for the fast detection of carbapenemase-producers have become commercially available. We evaluated a novel multiplex ICT for the rapid detection of OXA-48, KPC, NDM and VIM carbapenemases.
Methodology. One hundred well-characterized multidrug-resistant Enterobacteriaceae were analysed by RESIST-4 (Coris, BioConcept, Gembloux, Belgium). The reference standard included confirmation at the molecular level at the German National Reference Laboratory for multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany).
Results/Key findings. The tested ICT was highly reliable, showing 100 % sensitivity and 100 % specificity.
Conclusion. As it is fast, easy to perform and has few technical requirements, the ICT represents a good opportunity to improve turnaround times and patient care for the clinical microbiology laboratory.
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Host and pathogen factors in Klebsiella pneumoniae upper urinary tract infections in renal transplant patients
Purpose . To analyse the role of virulence factors (VFs) and host in Klebsiella pneumoniae upper urinary tract infections (UTIs) in renal transplant (RTx) recipients.
Methodology. Clinical and demographic data were registered prospectively. Phylogenetic background of K. pneumoniae isolates was analysed by PCR melting profiles (MP) and the following VFs genes: fimH-1, uge, kpn, ycfM, mrkD, rmpA, magA, hlyA, cnf-1, irp-1, irp-2, fyuA, entB, iutA, iroN by PCR.
Results. We studied urine cultures and clinical data from 61 episodes of K. pneumoniae UTI in 54 RTx recipients. There were 32 cases of AB (53%), 10 cases of lower UTI (16%), 19 cases of AGPN (31%), including six cases of bacteraemia. In total, 74 % of strains were extended-spectrum beta-lactamase+, and there were two carbapenemase-producing strains. PCR MP typing showed a diverse population with 52 different genetic profiles of K. pneumoniae . Analysis of the DNA profiles indicated 45 unrelated, unique genotypes and 7 related (16 isolates from 15 patients) genotypes. Urine flow impairment emerged as an independent predictor of K. pneumoniae upper UTIs (OR 14.28, CI 2.7–75.56, P 0.002), while we did not find any association between the profile of VFs and developing upper UTIs. The prevalence of the uge gene was lower in RTx patients on everolimus when compared to isolates from patients not receiving mTOR inhibitors (33.3 % vs 82.8 % P<0.05).
Conclusions . K. pneumoniae upper UTI may be a marker of urine flow impairment. Bacterial VFs could not discriminate between upper and lower UTIs. However, immunosuppression may influence the selection of particular VFs.
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- Disease, Diagnosis and Diagnostics
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Stool PCR may not be a substitute for enrichment culture for the detection of salmonella
More LessPurpose. Polymerase chain reaction (PCR) is increasingly being used to detect enteric pathogens and is currently NICE’s recommended practice. We wished to evaluate the performance characteristics of PCR for the detection of salmonella in consecutive stool samples in a real-world setting, compared to the gold standard of enrichment culture.
Methodology. We performed a prospective study over 9 months in which the PCR and culture results for salmonella were scrutinized for all stool samples sent to the laboratory. All stool samples underwent selenite enrichment culture for salmonella with confirmation being obtained using the API 10S and serotyping. Samples also underwent PCR using the BD MAX Enteric Bacterial Panel. The sensitivity and specificity of PCR in detecting salmonella were compared to those of enrichment culture.
Results. Six thousand three hundred and seventy-two stool culture and PCR pairs from 5619 patients were analysed. The prevalence of salmonella was found to be 1.2 %. The sensitivity, specificity, positive predictive value and negative predictive value of PCR versus culture were 89 % (67/75), 99.8 % (6286/6297), 86 % (67/78) and 99.9 % (6286/6294), respectively.
Conclusion. Enrichment culture is significantly more sensitive than PCR using the BD MAX Enteric Bacterial Panel for detecting salmonella in stool. Where PCR testing is used for the detection of enteric pathogens, we recommend that enrichment culture for salmonella be continued in parallel, unless the PCR method is shown to be at least as sensitive as culture.
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Performance of bioMérieux Lowenstein–Jensen slopes in plastic tube packaging, compared to existing phenotypic methods, for efficient recovery of the Mycobacterium tuberculosis complex
Purpose. Lowenstein–Jensen (LJ) medium used for cultivating Mycobacterium tuberculosis (MTB) is marketed in glass packaging. Breakage of glass slope is a major biosafety risk, especially during processing and storage, which gets magnified in large laboratories. We evaluated the performance of new bioMérieux (bMx) LJ slopes in plastic packaging, compared to bMx glass LJ medium and Becton Dickinson Mycobacterial Growth Indicator Tube (MGIT), for MTB recovery.
Methodology. Consecutive pulmonary/extra-pulmonary samples (n=240) were processed using routine methods of decontamination, inoculation and incubation.
Results. Plastic LJ slopes detected all 213 true-positive cases. The mean time-to-growth detection was 17.97 days for plastic LJ slopes, compared to 18.08 and 13.53 days for glass LJ slopes and MGIT, respectively. No statistically significant difference was observed between the two LJ slopes (P< 0.05). Both LJ slopes had a sensitivity and specificity of 100%, with respect to MGIT.
Conclusion. Plastic LJ slopes are a good alternative to the traditional glass slopes. The medium quality did not differ with the packaging material. Increased surface area of these slants allowed enhanced growth, and the clear plastic material allowed accurate recording of growth. The wide mouth of these containers eased inoculation. Increased biosafety, by elimination of breakage risk, is the biggest advantage of this modification.
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- Microbiome and Microbial Ecology in Health
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Effects of Eclipta prostrata on gut microbiota of SAMP6 mice with osteoporosis
More LessPurpose. Traditional Chinese medicine (TCM) has been extensively studied for its preventive and treatment properties toward osteoporosis (OP). Pharmacological studies have shown that TCM Eclipta prostrata induce anti-OP effects. Considering the growing evidence demonstrating that gut microbiota (GM) is related to OP, we aimed to study the GM-dependent function and mechanism of E. prostrata for preventing OP in mice.
Methodology. Bone micro-structure was obtained using micro-computed tomography (micro-CT) and bone-relating factors were detected by molecular biological test. High-throughput sequencing of the 16S rRNA V4 region was performed for GM diversity analysis. Growth effects of E. prostrata on potential targeted strains Lactobacillus and Lactococcus were investigated by in vitro bacterial assay. By feeding Lactobacillus and Lactococcus in mice, GM and bone condition were analysed.
Results. Bone micro-structure was significantly improved by E. prostrata with a potential mechanism of inhibiting osteoclast, increasing the number of osteoblasts and regulating the dynamic balance of bone absorption and formation. Sequencing results indicated that E. prostrata altered the bacterial community. The abundance of bacteria genera Lactobacillus and Lactococcus was markedly decreased in individuals with OP and positively correlated with high dose of E. prostrata. GM of the low-dose E. prostrata-fed group did not significantly differ from that of the chow-fed OP group, which was consistent with bone structure test results. Moreover, E. prostrata could promote Lactobacillus and Lactococcus growth in vitro. GM was altered and bone condition was improved via bacterial feeding in vivo.
Conclusion. Our findings suggested that E. prostrata might be a novel therapy for OP prevention by targeting GM.
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Characterization of intestinal Escherichia coli isolated from calves with diarrhea due to rotavirus and coronavirus
Purpose. To address more information about changes in commensal Escherichia coli during virus intestinal infection, we characterized 30 faecal E. coli isolates from calves (21 to 60 days old) with diarrhea due to rotavirus and coronavirus, which received, before diagnosis, tetracycline, gentamicin and enrofloxacin drugs.
Methodology. Clermont’s phylogenetic classification; presence of genes for curli, cellulose, fimbriae (F4, F5, F6, F18, F41); and antimicrobial susceptibility were used to characterize the isolates. Disk diffusion technique and PCR were used as methodologies.
Results. E. coli isolates from calves with diarrhea were phylogenetically classified as B1 (70%, 21/30), B2 (3.33%, 1/30), C (3.33%, 1/30), D (3.33%, 1/30), E (13.33%, 4/30) and unknown (6.7 %; 2/30), whereas E. coli isolates from the control group were classified only as B1 (83.3%, 25/30), E (10 %; 3/30) and unknown (6,7 %; 2/30). E. coli isolates from calves with diarrhea showed a much higher resistance profile with 16 (53.3%) multiresistant isolates. Only isolates (30%-9/30) from diarrheic calves were also positive for fimbriae, specifically 16.7% (5/30) for F5 and 13.3% (4/30) for F18.
Conclusion. To sum up, E. coli isolates from calves with diarrhea showed differences in relation to the control group, confirming changes in commensal E. coli during virus intestinal infection. It can be emphasized that some care should be taken to manage diarrheic calves: the pathological agent must be diagnosed prior to treatment; antibacterial treatment should be with antimicrobials with a different mechanism of action; and finally, treated animals should be maintained separately from others because they can carry micro-organisms with a resistant profile.
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- Molecular and Microbial Epidemiology
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Parechovirus A3 (PeV-A3)-associated myalgia/myositis occurs irrespective of its genetic cluster: a longitudinal molecular epidemiology of PeV-A3 in Yamagata, Japan between 2003 and 2016
No longitudinal molecular epidemiology of parechovirus A3 (PeV-A3) over a decade is available and PeV-A3-associated myalgia/myositis has been reported only in Japan. Thus, we aimed to clarify the longitudinal molecular epidemiology of PeV-A3 with a major focus on the strains detected from PeV-A3-associated myalgia/myositis cases. We performed sequence and phylogenetic analysis for the VP1 region of PeV-A3 strains in Yamagata, Japan, between 2003 and 2016. The phylogenetic analysis indicated that PeV-A3 strains caused PeV-A3-associated myalgia/myositis as well as a variety of infectious diseases, ranging from mild to severe, in subjects ranging from neonates to adults, irrespective of genetic cluster or variations. PeV-A3 strains are causative agents of a variety of human diseases, irrespective of their genetic cluster. Furthermore, we consider that PeV-A3-associated myalgia/myositis may occur, not only in Japan, but also in other countries, as closely related PeV-A3 strains have been circulating around the world.
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Whole-genome sequencing of the rare Salmonella enterica serovar Anfo isolated from food handlers
Field investigations were conducted after a small cluster of food poisoning involving six cases was reported. While no stool samples were available from the cases for microbiological testing, Salmonella species was found to be present in the stools of food handlers with gastroenteritis symptoms. Four Salmonella isolates recovered from the food handlers were retrospectively investigated at the genome level using whole-genome sequencing (WGS). WGS showed that S. Anfo (antigenic formulae 39:y:1,2), a rarely isolated serovar, caused infections in the food handlers. S. Anfo analysed in this study contained virulence factors required for causing disease. They did not contain any antibiotic resistance genes or plasmid. The epidemiologically related isolates differed to each other by a maximum of one single nucleotide polymorphism. WGS was useful in identifying rare Salmonella serovars and it is potentially more cost-effective than traditional serotyping methods. It can also confidently group epidemiologically related isolates belonging to S. Anfo.
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New in vitro model evaluating antiseptics’ efficacy in biofilm-associated Staphylococcus aureus prosthetic vascular graft infection
Purpose . To develop a new in vitro model of prosthetic vascular graft infection (PVGI) and evaluate antimicrobial and biofilm-disrupting efficacy of 0.1% octenidine dihydrochloride, 10% povidone-iodine and 0.02% chlorhexidine digluconate against biofilm-producing Staphylococcus aureus ( S. aureus ).
Methodology . The effect of antiseptics on the microscopic integrity and antimicrobial effect on S. aureus biofilms was tested by growing biofilms on glass coverslips, in the modified Lubbock chronic wound pathogenic biofilm (LCWPB) model and on the surface of vascular grafts using qualitive and quantitative methods as well as by scanning electron microscopy (SEM).
Results . Chlorhexidine worked best on destroying the integrity of S. aureus biofilms (P=0.002). In the LCWPB model, octenidine and povidone-iodine eradicated all S. aureus colonies (from 1.79 × 109 c.f.u. ml−1 to 0). In the newly developed PVGI model, the grafts were successfully colonized with biofilms as seen in SEM images. All antiseptics demonstrated significant antimicrobial efficacy, decreasing colony counts by seven orders of magnitude (P=0.002). Octenidine was superior to povidone-iodine (P=0.009) and chlorhexidine (P=0.041).
Conclusion . We implemented an innovative in vitro model on S. aureus biofilms grown in different settings, including a clinically challenging situation of PVGI. The strongest antimicrobial activity against S. aureus biofilms, grown on prosthetic vascular grafts, was showed by 0.1% octenidine dihydrochloride. We suggest that combinational therapy of antiseptics between chlorhexidine with either povidone-iodine or octenidine dihydrochloride should be tested in further experiments. Despite the need of further studies, our findings of these in vitro experiments will assist the management of vascular graft infection in clinical cases.
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- Pathogenesis, Virulence and Host Response
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Impact of HPV infection on gene expression and methylation in oral cancer patients
Purpose. The current study aimed to examine the association between head and neck squamous cell carcinoma (HNSCC) and infection with different human papillomavirus virus (HPV) subtypes, including analysis of promoter methylation of several genes (APC, CDKN2A, MGMT, CDH1 and TIMP3) and the correlation with their mRNA expression in tumours and surgical margins.
Methodology. In 47 patients with a primary tumour of the oral cavity, HPV detection and identification of 33 subtypes was performed after previous DNA isolation using a GenoFlow HPV Array Test Kit.
Results. Fifteen patients (31.92 %) were HPV [+] and the following HPV types were detected: 16 (46.67 %), 18 (6.67 %) and 43/44 (40 %). This study is the first to describe HPV 43/44 subtypes in HNSCC in a Polish population. We noted no clinical significance of HPV [+] HNSCC compared to HPV [−], however, this differed among HPV subtypes. CDKN2A promoter methylation was more frequent in HPV-16/18 patients compared to HPV43/44 patients, but there was no difference in gene expression level between HPV [+] and [−] patients.
Conclusion. We detected HPV infection in 31.92 % of oral cancer cases. HPV 16, along with HPV 43/44, were the most frequent subtypes. Knowledge of HPV [+] HNSCC biology may be useful in establishing the prognosis and developing novel therapies in future.
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Agmatine accumulation by Pseudomonas aeruginosa clinical isolates confers antibiotic tolerance and dampens host inflammation
More LessPurpose. In the cystic fibrosis (CF) airways, Pseudomonas aeruginosa undergoes diverse physiological changes in response to inflammation, antibiotic pressure, oxidative stress and a dynamic bioavailable nutrient pool. These include loss-of-function mutations that result in reduced virulence, altered metabolism and other phenotypes that are thought to confer a selective advantage for long-term persistence. Recently, clinical isolates of P. aeruginosa that hyperproduce agmatine (decarboxylated arginine) were cultured from individuals with CF. Sputum concentrations of this metabolite were also shown to correlate with disease severity. This raised the question of whether agmatine accumulation might also confer a selective advantage for P. aeruginosa during chronic colonization of the lung.
Methodology and Results. We screened a library of P. aeruginosa CF clinical isolates and found that ~5 % of subjects harboured isolates with an agmatine hyperproducing phenotype. Agmatine accumulation was a direct result of mutations in aguA, encoding the arginine deiminase that catalyses the conversion of agmatine into various polyamines. We also found that agmatine hyperproducing isolates (aguA-) had increased tolerance to the cationic antibiotics gentamicin, tobramycin and colistin relative to their chromosomally complemented strains (aguA+). Finally, we revealed that agmatine diminishes IL-8 production by airway epithelial cells in response to bacterial infection, with a consequent decrease in neutrophil recruitment to the murine airways in an acute pneumonia model.
Conclusion. These data highlight a potential new role for bacterial-derived agmatine that may have important consequences for the long-term persistence of P. aeruginosa in the CF airways.
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The effects of iclaprim on exotoxin production in methicillin-resistant and vancomycin-intermediate Staphylococcus aureus
More LessPurpose. Extracellular protein toxins contribute to the pathogenesis of Staphylococcus aureus infections. The present study compared the effects of iclaprim and trimethoprim – two folic acid synthesis inhibitors – with nafcillin and vancomycin on production of Panton–Valentine leukocidin (PVL), alpha haemolysin (AH) and toxic-shock syndrome toxin I (TSST-1) in methicillin-resistant and vancomycin-intermediate S. aureus (MRSA and VISA, respectively).
Methodology. Northern blotting and RT-PCR were used to assess gene transcription; toxin-specific bioassays were used to measure protein toxin production.
Results. As shown previously, sub-inhibitory concentrations (sub-MIC) of nafcillin increased and prolonged MRSA toxin gene transcription and enhanced PVL, TSST-1 and AH production. Sub-inhibitory doses of iclaprim and trimethoprim delayed maximal AH gene (hla) transcription and suppressed AH production; both drugs delayed, but neither reduced, maximal TSST-1 production. Trimethoprim significantly increased lukF-PV expression and PVL production compared to both untreated and iclaprim-treated cultures. Higher concentrations of iclaprim and trimethoprim markedly suppressed MRSA growth, mRNA synthesis and toxin production. In VISA, iclaprim, vancomycin and nafcillin variably increased tst and hla expression, but only nafcillin increased toxin production. Despite its ability to increase hla expression, iclaprim was the most potent inhibitor of AH production.
Conclusions. We conclude that, due to its ability to suppress toxin production, iclaprim should be effective against severe staphylococcal infections caused by toxin-producing MRSA and VISA strains, especially given its ability to concentrate at sites of infection such as skin and skin structures and the lung.
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Heat-treated Lactobacillus plantarum increases the immune responses through activation of natural killer cells and macrophages on in vivo and in vitro models
More LessPurpose. Recently, Lactobacillus plantarum (nF1) has been reported to have immune-enhancing effects in an immunosuppressed-animal model. Natural killer (NK) cells and macrophages play important roles in the immune responses. However, immunomodulatory effects of heat-treated Lactobacillus plantarum -nF1 (hLp-nF1) on the activation of NK cells and macrophages have not been elucidated.
Methodology. We assessed whether hLp-nF1 could elevate the activation of NK cells and macrophages using cyclophosphamide (CP)-induced immunosuppressed BALB/c mice and RAW 264.7 macrophages. A nitric oxide (NO) assay, enzyme-linked immunosorbent assay, Western blot analysis and NK cell activity assay were used to examine the effects of hLp-nF1 on the immune enhancement.
Results/Key findings. Administration of hLp-nF1-elevated NK cell activities and serum levels of TNF-α, IL-2, and IL-12 in CP-induced immunosuppressed mice. In RAW 264.7 macrophages, treatment with hLp-nF1 increased the production of NO and expression of inducible NO synthase. Simultaneously, hLp-nF1 increased the production of TNF-α, IL-2, and IL-6 from RAW 264.7 cells. Finally, hLp-nF1 induced activation of nuclear factor-κB and phosphorylation of IκBα.
Conclusion . We identified that hLp-nF1 has an immune-enhancing effect through the activation of NK cells and macrophages. Therefore, these findings suggest that hLp-nF1 would be helpful to enhance the immunity.
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- Prevention, Therapy and Therapeutics
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Minimum 5 years’ follow-up after gentamicin- and clindamycin-loaded PMMA cement in total joint arthroplasty
More LessPurpose. Due to numerous advantages of the combination of clindamycin and gentamicin in polymethylmethacrylate (PMMA) cement, promising preliminary results have been reported. However, there are no data that analyse mid-term outcomes.
Methodology.This pilot study included patients who experienced 5 years of follow-up and who were treated with gentamicin- and clindamycin-loaded (G+C) PMMA cement. They were divided into two groups: (1) the periprosthetic joint infection (PJI) comprising patients who underwent a one-stage exchange, and (2) aseptic group comprising patients who underwent aseptic revision or primary arthroplasty procedures, but were considered to be high-risk patients for infection. We evaluated the rate of septic and aseptic revision arthroplasty with a minimum of 5-year follow-up.
Results/Key findings. A total of 32 patients in both groups were included. Eighteen patients belonged to the PJI-group and 14 belonged to the aseptic group. There was no reinfection among the patients of the PJI group. Infection was prevented in the aseptic group, including patients with a history of PJI or at higher risk of infection. No patient underwent an exchange of the cemented prosthesis at the 5-year follow-up [72-82 months, standard deviation (sd)=3.3].
Conclusions. The local use of G+C bone cement during septic and aseptic revision arthroplasty, was associated with a high success rate for the eradication of infection following one-stage septic exchange, and with prevention of infection in high-risk patients.
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Incorporation of CD40 ligand or granulocyte-macrophage colony stimulating factor into Hantaan virus (HTNV) virus-like particles significantly enhances the long-term immunity potency against HTNV infection
Purpose. Hantavirus infections cause severe haemorrhagic fever with renal syndrome (HFRS) in humans and are associated with high fatality rates. In 2017, numerous outbreaks were reported in China and Germany. This represents a significant public-healthcare issue with no effective HFRS vaccines that offer a long-term immune response. In this study, we investigated the long-term humoral and cellular immune responses and protective immunity of Hantaan virus (HTNV) granulocyte-macrophage colony stimulating factor (GM-CSF) and CD40 ligand (CD40L) virus-like particles (VLPs) in mice.
Methodology. GM-CSF and CD40L VLPs were constructed via co-transfection of pCI-S and pCI-M-CD40L, and pCI-S and pCI-M-GM-CSF, into dihydrofolatereductase (dhfr)-deficient Chinese hamster ovary cells, respectively. Mice were immunized with HTNV VLPs 2 weeks apart. The animals were challenged 6 months after immunization. Specific and neutralizing antibodies were assessed by ELISA; IFN-γ was measured by enzyme-linked immunospot (ELISpot) assay and effectiveness by cytotoxic T lymphocyte (CTL) cytotoxicity assays. Nucleic acid loads of HTNV were tested by quantitative real-time PCR and viral antigen was detected via indirect ELISA. Pathological alterations were detected via haematoxylin–eosin staining.
Results. GM-CSF and CD40L VLPs provided stable, long-term protection with a high titre of neutralizing antibody in mice 6 months after immunization. Furthermore, VLPs increased HTNV-specific cellular immune responses via higher expression of IFN-γ and CTL responses. HTNV challenge assay results showed long-term protection against HFRS. No significant pathological alteration was observed in the organs of mice after immunization.
Conclusion. This is, to the best of our knowledge, the first report demonstrating the long-term potency of HTNV VLP vaccines against HTNV infection and offers new insights into HTNV vaccine development.
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In vitro activities of daptomycin combined with fosfomycin or rifampin on planktonic and adherent linezolid-resistant isolates of Enterococcus faecalis
Purpose. This study aimed to explore daptomycin combined with fosfomycin or rifampin against the planktonic and adherent linezolid-resistant isolates of Enterococcus faecalis .
Methodology. Four linezolid-resistant and four linezolid-sensitive isolates of E. faecalis which formed biofilms were collected for this study. Biofilm biomasses were detected by crystal violet staining and the adherent cells in the mature biofilms were quantified by c.f.u. determination.
Results. Daptomycin alone, or combined with fosfomycin or rifampin (4×MIC) demonstrated bactericidal activities on the planktonic cells, and daptomycin combined with fosfomycin killed more planktonic cells (at least 1−log10 c.f.u. ml−1) than daptomycin or fosfomycin alone. Daptomycin alone (16×MIC) showed anti-biofilm activities against the mature biofilms and bactericidal activities on the adherent cells, while daptomycin combined with fosfomycin (16×MIC) demonstrated significantly more anti-biofilm activities than daptomycin or fosfomycin alone and effectively killed the adherent cells in the mature biofilms. The high concentration of daptomycin (512 mg l−1 ) combined with fosfomycin indicated more bactericidal activities on the adherent cells and more anti-biofilm activities against the mature biofilms than daptomycin 64 mg l−1 (16×MIC) combined with fosfomycin. The addition of rifampin increased the anti-biofilm and bactericidal activities of daptomycin against the mature biofilms and the adherent cells of two isolates, however, which was not observed in other isolates.
Conclusions. Daptomycin combined with fosfomycin demonstrated better effect on the planktonic and adherent linezolid-resistant isolates of E. faecalis than daptomycin or fosfomycin alone. The role of rifampin in the treatment of E. faecalis isolates is discrepant and needs more studies.
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Volumes and issues
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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Volume 70 (2021)
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Volume 69 (2020)
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Volume 68 (2019)
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Volume 67 (2018)
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Volume 66 (2017)
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Volume 65 (2016)
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Volume 64 (2015)
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Volume 63 (2014)
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Volume 62 (2013)
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Volume 61 (2012)
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Volume 60 (2011)
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Volume 59 (2010)
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Volume 58 (2009)
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Volume 57 (2008)
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Volume 56 (2007)
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Volume 55 (2006)
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Volume 54 (2005)
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Volume 53 (2004)
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Volume 52 (2003)
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Volume 51 (2002)
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Volume 50 (2001)
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Volume 49 (2000)
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Volume 48 (1999)
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Volume 47 (1998)
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Volume 46 (1997)
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Volume 45 (1996)
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Volume 44 (1996)
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Volume 43 (1995)
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Volume 42 (1995)
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Volume 41 (1994)
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Volume 40 (1994)
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Volume 39 (1993)
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Volume 38 (1993)
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Volume 37 (1992)
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Volume 36 (1992)
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Volume 35 (1991)
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Volume 34 (1991)
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Volume 33 (1990)
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Volume 32 (1990)
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Volume 31 (1990)
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Volume 30 (1989)
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Volume 29 (1989)
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Volume 28 (1989)
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Volume 27 (1988)
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Volume 26 (1988)
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Volume 25 (1988)
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Volume 24 (1987)
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Volume 23 (1987)
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Volume 22 (1986)
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Volume 21 (1986)
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Volume 20 (1985)
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Volume 19 (1985)
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Volume 18 (1984)
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Volume 17 (1984)
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Volume 16 (1983)
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Volume 15 (1982)
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Volume 14 (1981)
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Volume 13 (1980)
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Volume 12 (1979)
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Volume 11 (1978)
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Volume 10 (1977)
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Volume 9 (1976)
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Volume 8 (1975)
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Volume 7 (1974)
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Volume 6 (1973)
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Volume 5 (1972)
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Volume 4 (1971)
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Volume 3 (1970)
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Volume 2 (1969)
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Volume 1 (1968)