To evaluate the accuracy and reproducibility of antimicrobial susceptibility testing methods in Burkholderia cepacia complex (BCC).
Minocycline, ciprofloxacin, trimethoprim/sulphamethoxazole, ...meropenem, ceftazidime and chloramphenicol were tested against 155 BCC strains using broth microdilution at 35 ± 1°C (BMD35) in triplicate, then BMD at 30 ± 1°C (BMD30), agar dilution at 30°C and 35°C (AD30 and AD35), gradient strip (GS) and EUCAST standardized disc diffusion (DD) testing methods once.
BMD35 reproducibility ranged from 70% to 84.5% for all agents. Correlations of MICs from BMD35 with BMD30 ranged from 63% to 85%, with AD35 from 32.9% to 87% and with GS methods from 36% to 83.9%. Essential agreement (EA) of MICs by GS with BMD35 ranged from 62.6% (trimethoprim-sulphamethoxazole) to 83.9% (minocycline). EA of EUCAST DD zone diameters using CLSI breakpoint criteria was between 85.8% and 97.4%, however Very Major Errors (VME) for trimethoprim/sulphamethoxazole were 31%.
BMD at 35 ± 1°C was poorly reproducible for most agents and no method showed acceptable performance. Of particular concern were the GS results. Although this is the most commonly used method for determining MICs in laboratories, there was poor correlation with BMD35 for meropenem and trimethoprim/sulphamethoxazole. EUCAST DD correlated poorly with BMD35 MICs. This study confirms that no susceptibility method is capable of providing reproducible and accurate MICs when testing BCC.
Lateral flow tests (LFTs) have been used to screen for SARS-CoV2 in Wales since January 2021. Between May and August 2021, adult care home staff policy was for biweekly Innova LFT and weekly ...Polymerase Chain Reaction (PCR) testing while asymptomatic. We estimated test performance of LFTs conducted in adult care home staff using PCR tests as a reference standard. Test results from surveillance data were matched by individual where both LFT and PCR were taken on the same day. We calculated sensitivity, specificity, positive and negative predictive values, and agreement using Matthew's correlation coefficient. Ct values of positive PCR results were compared by matched LFT result. Analysis was conducted using R v4.1.3. We analysed 115,593 test pairs, 499 (0.43%) of which were PCR positive. Median age was 48 (IQR: 22) and 85.00% of the study population were female. Test result agreement was 99.59% (95%CI 99.55-99.63; MCC: 0.38, p<0.001). Sensitivity and specificity were 25.65% (95%CI 22.02-29.67) and 99.91% (95%CI 99.89-99.93), respectively. PPV was 55.90% (95%CI 49.42-62.17) and NPV was 99.68% (95%CI 99.64-99.71). Crude Ct values were significantly lower in positive PCR tests matched to a positive LFT compared to a negative LFT. Specificity and negative predictive value were high in an asymptomatic population of care home staff indicating this test is an effective tool for identifying cases of SARS-CoV-2 infection during periods of high prevalence where transmission is likely, due to the presence of high viral loads. Positive predictive value results are lower than existing literature yet should be considered in light of the asymptomatic study population and low prevalence (under 1%) at the time most of these tests were conducted. Performance improved at times of higher prevalence during the study. These results suggest that whilst lateral flow tests are effective for identifying SARS-COV-2 infections with high viral loads, they are not effective at identifying cases with a low viral load. When an LFT provides a negative result, false negatives should be considered and additional diagnostic tests performed.
Antibiotic resistance is a significant global public health concern. Uropathogenic Escherichia coli sequence type (ST)131, a widely prevalent multidrug-resistant clone, is frequently associated with ...bacteraemia. This study investigates third-generation cephalosporin resistance in bloodstream infections caused by E. coli ST131. From 2013-2014 blood culture surveillance in Wales, 142 E. coli ST131 genomes were studied alongside global data. All three major ST131 clades were represented across Wales, with clade C/H30 predominant (n = 102/142, 71.8%). Consistent with global findings, Welsh strains of clade C/H30 contain β-lactamase genes from the bla
group (n = 65/102, 63.7%), which confer resistance to third-generation cephalosporins. Most Welsh clade C/H30 genomes belonged to sub-clade C2/H30Rx (58.3%). A Wales-specific sub-lineage, named GB-WLS.C2, diverged around 1996-2000. An introduction to North Wales around 2002 led to a localised cluster by 2009, depicting limited genomic diversity within North Wales. This investigation emphasises the value of genomic epidemiology, allowing the detection of genetically similar strains in local areas, enabling targeted and timely public health interventions.
The BSAC Standing Committee on Antimicrobial Susceptibility Testing is one of several European national breakpoint committees that agreed in 2002 to harmonize clinical MIC breakpoints. The process of ...harmonization has since been completed for commonly used agents, and breakpoints for new agents have been set by EUCAST in accordance with a procedure defined by the EMA. EUCAST breakpoints have now been adopted by a large majority of laboratories in Europe. BSAC implemented the EUCAST breakpoints in its own disc diffusion susceptibility testing method as harmonized breakpoints were agreed to over the years. Since the development of the EUCAST disc diffusion method, several countries with their own disc diffusion methods have switched to the EUCAST method, and BSAC will replace support of its own disc diffusion method with support for the EUCAST method from January 2016. The EUCAST breakpoints are also available in automated systems. The harmonized breakpoints and methods will help to avoid different reports of susceptibility for the same isolate in different countries and enable more reliable comparison of resistance rates in surveillance studies in different countries.
Infectious gastrointestinal disease is caused by a diverse array of pathogens, and is a challenging syndrome to correctly diagnose and manage. Conventional laboratory diagnostic methods are often ...time-consuming and frequently suffer from low detection rates. Two commercial multiplex nucleic acid amplification tests Luminex xTAG Gastrointestinal Pathogen Panel (GPP) and Savyon Diagnostics Gastrointestinal Infection Panel (GIP) were applied to 1000 stored diarrhoeal clinical stool samples. The Luminex xTAG GPP and Savyon GIP detected Campylobacter in 42/44 and 44/44 culture-positive samples, Salmonella in 4/4 and 3/4 culture-positive samples, Shigella in 1/1 culture-positive sample, Clostridium difficile toxin in 32/35 ELISA-positive samples, and Giardia in 6/6 wet-preparation-microscopy-positive samples, respectively. When the Luminex GPP assay was used concurrently with conventional methods for 472 clinical samples, it detected Campylobacter in 22/22 culture-positive samples, Salmonella in 1/1 culture-positive sample, Clostridium difficile toxin in 14/14 ELISA-positive samples and Giardia in 4/4 wet-preparation-microscopy-positive samples. The pathogen/toxin detection rate for conventional methods in both sample sets was <10%. The Luminex xTAG GPP detection rate was 24.8% in the stored samples and 32.6% in the concurrently tested samples. The Savyon GIP detection rate was 22.5%. From stored samples, 2.4% of Luminex xTAG GPP detections and 3.1% of Savyon GIP detections could not be confirmed using alternative nucleic acid amplification tests. Enhanced detection rates resulted from increased detection of pathogens routinely sought using conventional methods and were also due to ascertainment of micro-organisms that current testing strategies do not diagnose. Use of multiplex nucleic acid amplification tests will allow clinical laboratories to diagnose infectious gastroenteritis in more patients with diarrhoeal disease by increasing the sensitivity of pathogen detection and by reducing the selective bias of current strategies. The clinical and economic impact of these results warrants further investigation.
Susceptibility testing of bacteria is one of the most important tests performed in a clinical microbiology laboratory. Improvements in laboratory techniques, especially the move towards standardized ...susceptibility testing, has provided better consistency and accuracy of testing. When used in conjunction with the most recently developed interpretative criteria, the result is better prediction of the outcome of antimicrobial therapy for infected patients. Throughout the last four decades this Journal has published numerous articles evidencing improvements and new techniques, a valuable source of information for microbiology laboratories.
Glycopeptide-intermediate Staphylococcus aureus (GISA) and heterogeneous GISA (hGISA) strains are notoriously difficult to detect in the diagnostic laboratory. The clinical importance of GISA, and ...particularly hGISA, will only be obvious when a definitive detection method is available. A few novel GISA and hGISA detection methods have been proposed; however, their validity has never been tested on a significant scale and in different laboratories. This study compares three screening methods for detecting GISA and hGISA strains in 12 laboratories, using a blind panel of 48 strains with known glycopeptide susceptibilities. The three screening methods used were brain heart infusion agar with 6 mg/liter vancomycin (BHIA6V) (CDC/CLSI), Mueller-Hinton agar with 5 mg/liter teicoplanin (MHA5T) (European Antimicrobial Resistance Surveillance System EARSS), and the macrodilution method Etest (MET) (EARSS), with population analysis profile-area under the curve analysis as the gold standard. Sensitivity and specificity were highest for MHA5T and MET, which identified 82.5% and 85.9% of strains, respectively. BHIA6V had poor sensitivity, particularly for hGISA (11.5% of strains were detected), and gave the largest interlaboratory variation in performance. MET exhibited the least interlaboratory variation. It is essential that laboratories use appropriate methods to detect GISA/hGISA strains so that the prevalence and clinical importance of these strains can be assessed properly.
The blaNDM-1 gene is associated with extensive drug resistance in Gram-negative bacteria. This probably spread to Enterobacteriaceae from Acinetobacter spp., and we characterized plasmids associated ...with blaNDM-1 in Acinetobacter spp. to gain insight into their role in this dissemination. Four clinical NDM-1-producing Acinetobacter species strains from India and Pakistan were investigated. A plasmid harboring blaNDM-1, pNDM-40-1, was characterized by whole-genome sequencing of Acinetobacter bereziniae CHI-40-1 and comparison with related plasmids. The presence of similar plasmids in strains from Pakistan was sought by PCR and sequencing of amplicons. Conjugation frequency was tested and stability of pNDM-40-1 investigated by real-time PCR of isolates passaged with and without antimicrobial selection pressure. A. bereziniae and Acinetobacter haemolyticus strains contained plasmids similar to the pNDM-BJ01-like plasmids identified in Acinetobacter spp. in China. The backbone of pNDM-40-1 was almost identical to that of pNDM-BJ01-like plasmids, but the transposon harboring blaNDM-1, Tn125, contained two short deletions. Escherichia coli and Acinetobacter pittii transconjugants were readily obtained. Transconjugants retained pNDM-40-1 after a 14-day passage experiment, although stability was greater with meropenem selection. Fragments of pNDM-BJ01-like plasmid backbones are found near blaNDM-1 in some genetic contexts from Enterobacteriaceae, suggesting that cross-genus transfer has occurred. pNDM-BJ01-like plasmids have been described in isolates originating from a wide geographical region in southern Asia. In vitro data on plasmid transfer and stability suggest that these plasmids could have contributed to the spread of blaNDM-1 into Enterobacteriaceae.
We recently described a novel antimicrobial peptide, RTA3, derived from the commensal organism Streptococcus mitis, with strong anti-Gram-negative activity, low salt sensitivity, and minimal ...mammalian cell toxicity in vitro and in vivo. This peptide conforms to the positively charged, amphipathic helical peptide motif, but has a positively charged amino acid (Arg-5) on the nonpolar face of the helical structure that is induced upon membrane binding. We surmised that disruption of the hydrophobic face with a positively charged residue plays a role in minimizing eukaryotic cell toxicity, and we tested this using a mutant with an R5L substitution. The greatly enhanced toxicity in the mutant peptide correlated with its ability to bind and adopt helical conformations upon interacting with neutral membranes; the wild type peptide RTA3 did not bind to neutral membranes (binding constant reduced by at least 1000-fold). Spectroscopic analysis indicates that disruption of the hydrophobic face of the parent peptide is accommodated in negatively charged membranes without partial peptide unfolding. These observations apply generally to amphipathic helical peptides of this class as we obtained similar results with a peptide and mutant pair (Chen, Y., Mant, C. T., Farmer, S. W., Hancock, R. E., Vasil, M. L., and Hodges, R. S. (2005) J. Biol. Chem. 280, 12316–12329) having similar structural properties. In contrast to previous interpretations, we demonstrate that these peptides simply do not bind well to membranes (like those of eukaryotes) with exclusively neutral lipids in their external bilayer leaflet. We highlight a significant role for tryptophan in promoting binding of amphipathic helical peptides to neutral bilayers, augmenting the arsenal of strategies to reduce mammalian toxicity in antimicrobial peptides.
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