Purpose. Thymidine-dependent small-colony variants (TD-SCVs) are difficult to detect or test for antimicrobial susceptibility. We investigated the characteristics of clonal TD-SCVs of Escherichia coli, both with and without bla CTX-M-3, isolated from a patient.
Methodology. Mutation in the thyA gene was analysed by sequencing, and morphological abnormalities in the colonies and cells of the isolates were examined. Additionally, conjugational transfer experiments were performed to prove the horizontal transferability of plasmids harbouring resistance genes.
Results. The TD-SCVs contained a single nucleotide substitution in the thyA gene, c.62G>A, corresponding to p.Arg21His. Morphologically, their colonies were more translucent and flattened than those of the wild-type strain. In addition, cells of the TD-SCVs were swollen and elongated, sometimes with abnormal and incomplete divisions; a large amount of cell debris was also observed. Changing c.62G>A back to the wild-type sequence reversed these abnormalities. Conjugational transfer experiments showed that the TD-SCV of E. coli with bla CTX-M-3 failed to transfer bla CTX-M-3 to E. coli CSH2. However, the TD-SCV of E. coli without bla CTX-M-3 experimentally received the plasmid encoding bla SHV-18 from Klebsiella pneumoniae ATCC 700603 and transferred it to E. coli CSH2.
Conclusion. Mutation in the thyA gene causes morphological abnormalities in the colonies and cells of E. coli, as well as inducing thymidine auxotrophy. In addition, TD-SCVs horizontally transmit plasmids encoding resistance genes. It is important to detect TD-SCVs based on their characteristics because they serve as reservoirs of transferable antibiotic resistance plasmids.
Purpose. The microbiome from nursing home (NH) residents is marked by a loss in diversity that is associated with increased frailty. Our objective was to explore the associations of NH environment, frailty, nutritional status and residents’ age to microbiome composition and potential metabolic function.
Methodology. We conducted a prospective longitudinal cohort study of 23 residents, 65 years or older, from one NH that had four floors: two separate medical intensive floors and two floors with active elders. Residents were assessed using the mini nutritional assessment tool and clinical frailty scale. Bacterial composition and metabolic potential of residents' stool samples was determined by metagenomic sequencing. We performed traditional unsupervised correspondence analysis and linear mixed effect modelling regression to assess the bacteria and functional pathways significantly affected by these covariates.
Results/Key findings. NH resident microbiomes demonstrated temporal stability (PERMANOVA P=0.001) and differing dysbiotic associations with increasing age, frailty and malnutrition scores. As residents aged, the abundance of microbiota-encoded genes and pathways related to essential amino acid, nitrogenous base and vitamin B production declined. With increasing frailty, residents had lower abundances of butyrate-producing organisms, which are associated with increased health and higher abundances of known dysbiotic species. As residents became malnourished, butyrate-producing organisms declined and dysbiotic bacterial species increased. Finally, the microbiome of residents living in proximity shared similar species and, as demonstrated for Escherichia coli, similar strains.
Conclusion. These findings support the conclusion that a signature ‘NH’ microbiota may exist that is affected by the residents' age, frailty, nutritional status and physical location.
Objective. The aim of this study was to perform molecular characterization for and determine the antimicrobial susceptibility profiles of Clostridium difficile collected from hospitals during a 4-year period (2009–2013) in China.
Methods. Strains of toxigenic C. difficile were isolated from patients with diarrhoea, and this was followed by typing using multilocus sequence typing (MLST) and testing for susceptibility to 10 antimicrobials by using the E-test. The mechanisms of resistance to moxifloxacin, erythromycin, clindamycin and tetracycline were investigated by PCR.
Results. A total of 405 non-duplicate toxigenic C. difficile isolates were identified, while 31 sequence types (STs) were identified. A predominant type, ST-54, accounted for 20.2 % of the STs, followed by ST-35 (16.3 %) and ST-37 (13.6 %). We found that 6.2 % of the isolates were binary toxin genes-positive, and 83.7 % of these belonged to ST-5. All of the isolates demonstrated 100 % susceptibility to first-line Clostridium difficile infection (CDI) therapies (i.e. metronidazole and vancomycin), while the resistance rates varied for the other antibiotics tested. Two hundred and ninety three (72.3 %) isolates were susceptible to moxifloxacin. All 112 moxifloxacin-resistant isolates had mutations resulting in an amino acid substitution in gryA and/or gyrB. The ermB gene was detected in 86.7 % (241/278) of the erythromycin- and clindamycin-resistant isolates, while the tetM gene was present in 97.1 % (85/87) of the tetracycline-resistant isolates.
Conclusion. MLST typing revealed a wide variety of STs causing CDI, while ST-54 was the most common ST. All of the isolates were susceptible to metronidazole and vancomycin, while the resistance rates varied for the other antibiotics tested. There were no changes in the trends for the STs and antibiotic susceptibility profiles over 4 years.
Purpose. This study explored the prevalence and characteristics of Enterococcus faecalis biofilm formation by urinary tract infection (UTI) isolates in order to identify virulence factors associated with biofilm formation.
Methodology. A total of 113 E. faecalis isolates were collected from UTI patients in Shenzhen, China. The isolates were subjected to multilocus sequence typing based on housekeeping genes. Biofilms were detected by crystal violet staining and the expression levels of the E. faecalis genes were detected by quantitative real-time PCR.
Results/Key findings. The main sequence types (STs) were ST16 and ST179 with the ST16 isolates more likely to form strong biofilms than the ST179 isolates (P=0.008). Strong biofilm formation was more frequently detected in aggregation substance (agg)-positive (+) isolates than in negative (−) isolates (P=0.033). Biofilm formation was also more common in isolates containing enterococcal surface protein (esp), or cytolysin A (cylA)-positive (+) isolates than in isolates negative (−) for these virulence factors. Multivariate regression analysis indicated that cylA [odds ratio (OR), 7.143, P=0.012] was associated with weak biofilm formation, and that agg (OR, 4.471, P=0.004) was associated with strong biofilm formation. The expression of cylA was increased (8.75- to 23.05-fold) in weak biofilm, and the expression of agg was greatly elevated (11.99- to 439.10-fold) in strong biofilm isolates when compared to biofilm-negative isolates.
Conclusion. ST16 classification was positively associated with strong biofilm formation in E. faecalis as was agg, while cylA was associated with weak biofilm formation.
Purpose. Neisseria gonorrhoeae is a sexually transmitted bacterial pathogen that continues to evolve to become resistant to known antibiotics. In preparing for potential emergence, the Centers for Disease Control and Prevention recommends that clinical laboratories maintain or develop protocols to assess antibiotic susceptibly for this organism. This study examines the intra-laboratory variability of using the Etest method to provide consistent MIC values for N. gonorrhoeae and also compared the results of the Etest to known agar dilution MIC values.
Methodology. Clinical N. gonorrhoeae isolates, 100 paired duplicates, were tested by eight laboratories for antibiotic susceptibility to ceftriaxone, cefixime and azithromycin using Etest strips.
Results/Key findings. Overall, >80 % of the paired Etest MIC values were within one log2 dilution of the replicate. When compared to the agar dilution reference method, the cefixime Etest MIC values were consistently underreported by one dilution (seven laboratories) or two dilutions (one laboratory). The azithromycin Etest MIC values agreed 90.7 % with the agar dilution MIC values while the agreement with ceftriaxone was 90.9 %.
Conclusion. Overall, the Etest method yielded reproducible MIC values within each laboratory with the azithromycin and ceftriaxone MIC results consistent to the reference agar dilution method while the cefixime result tended to provide a lower MIC value.
Purpose. Macrolide susceptibility differs between subspecies in the Mycobacterium abscessus complex, likely due to differences in erm(41) sequevars. Patients with M. abscessus complex infection generally show poor clinical outcomes in response to antibiotic treatment. Here, the association between genotype and treatment outcome was investigated.
Methodology. We collected 69 isolates from 35 patients with non-cystic fibrosis bronchiectasis: 24 had M. abscessus complex lung disease and non-cystic fibrosis bronchiectasis, and 11 were colonized. Outcome analysis was performed in the 24 infected patients. Molecular analyses, including erm(41) and rrl sequencing, and variable-number tandem-repeat (VNTR) analysis of 69 isolates, from 24 infected and 11 colonized patients, were performed to elucidate the influence of genotype on antibiotic susceptibility.
Results. Among the 24 patients, 18 (14 infected with M. abscessus subsp. abscessus and 4 with M. abscessus subsp. massiliense) showed unfavourable outcomes; six (three infected with M. abscessus subsp. abscessus and three with M. abscessus subsp. massiliense) exhibited favourable outcomes. Patients with unfavourable outcomes showed acquired clarithromycin resistance (33.3 vs 0 %), mixed sequevars (38.9 vs 16.7 %) and differing VNTR patterns between initial and serial isolates (33.3 vs 16.7 %). In contrast, in the 11 colonized patients, M. abscessus subsp. abscessus C28 (sequevar 02) and M. abscessus subsp. massiliense were the most prevalent subspecies.
Conclusion. Patients infected with multiple sequevars and genotypes were more likely to exhibit treatment failure and/or recurrence. The precise identification of subspecies and analyses of mycobacterial characteristics may help to predict treatment outcomes in patients with M. abscessus complex lung disease.