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Objectives: Rapid antibiotic susceptibility testing (AST) for positive blood cultures can improve patient clinical outcomes if the time to an effective antimicrobial therapy is shortened. In this study, we tested the Quantamatrix dRAST system (QMAC-dRAST), a rapid AST system based on time-lapse microscopic imagery of bacterial colony formation in agarose. Methods: Evaluation of the QMAC-dRAST was performed from 250 monobacterial blood cultures including 130 Enterobacterales, 20 non-fermentative Gram-negative bacteria, 69 staphylococci and 31 enterococci. Blood cultures were recovered from anonymous patients or from spiking experiments to enrich our study with bacterial species and resistant strains. Categorical agreement (CA), minor errors (me), major errors (ME) and very major errors (VME) were calculated in comparison to the results obtained from the BD Phoenix™ M50. Discrepancies between the Phoenix™ M50 and QMAC-dRAST results were investigated using the gradient strip method. The repeatability and reproducibility performance of the QMAC-dRAST was assessed for 16 strains, each strain being tested five times from a spiked blood culture. Results: The overall CAs for Enterobacterales, non-fermentative Gram-negative bacteria, staphylococci and enterococci were 95.1%, 91.2%, 93.4% and 94.5%, respectively. The VME percentage was below 4% for all the groups except for staphylococci, which showed a VME rate of 7%. The median time to result was 6.7 h (range: 4.7–7.9). Repeatability and reproducibility assays showed a high reliability of AST results with best and worst ratios of 98.8% and 99.6% and 95.0% and 98.3%, respectively. Conclusions: The QMAC-dRAST is a fast and reliable system to determine AST directly from monobacterial blood cultures with a major TAT reduction compared to conventional AST testing.

Since the introduction of antibiotics, successive waves of Staphylococcus aureus clones occurred, each one having characteristic susceptibility pattern to antibiotics and virulence factors. We report here the results of a molecular epidemiological surveillance of methicillin-resistant S. aureus (MRSA) in French-speaking Switzerland between 2006 and 2020 showing the emergence and disappearance of clones known for their international dissemination, and the sporadic appearance of other international clones. Since 2012, a marked decrease in the incidence of cases attributable to the biology of the clones and to the control measures taken in the hospitals has been observed. These results highlight the importance of continuous surveillance in order to better assess the burden of this multi-resistant pathogen in our region.Depuis l’introduction des antibiotiques, des vagues successives de clones de

Toxigenic Clostridioides difficile is responsible for up to one third of post antibiotic diarrhea and for more than 95% of pseudomembranous colitis. Nowadays, diagnosis relies on the documentation of the presence of the toxin in stools by specific antigenic or PCR tests. Stool cultures have been mostly abandoned, leading to the absence of isolates for further epidemiological analyses. Aliquots of stool samples, frozen for up to two years, were thawed and inoculated onto commercial C. difficile media. Eighteen stools were recovered from patients hospitalized in the pediatric ward where at that time a chain of transmission was suspected. Eleven stools were recovered from patients hospitalized in a medical ward over a three months period with no suspected transmission event. Up to 16 characteristic colonies were isolates per culture. PCR of toxins genes and molecular typing by Double Locus Sequence Typing (DLST) were performed on these colonies. Whole genome multi locus sequence typing (wgMLST) was performed on selected isolates. Among the 29 stool specimens, no growth was observed for four stools and only one colony grew for one stool. Except the latter, all 16 colonies of the 24 stools showed identical toxin genes profiles than the original stool. However, variant DLST genotypes was observed within 20% of investigated stools. The majority of variants were single locus variant due to an IN/DEL of the repeat in one of the two DLST locus. Despite this variation, results of molecular typing overrule the putative transmission chain in the pediatric ward and revealed undetected chains of transmission in the medical ward. These results were confirmed with wgMLST. The developed protocol allows prospective and retrospective molecular and genomic epidemiological investigation of C. difficile infections for infection control purpose.

BACKGROUND: Campylobacter spp. are a frequent cause of gastroenteritis, presenting in some patients as an acute abdominal emergency. Here we describe the distinctive clinical characteristics of these patients. METHODS: We designed a retrospective, single-centre, observational study. Children and adolescents under 18 years of age who had positive stool cultures for Campylobacter spp. during the period between June 1, 2008 and May 31, 2016 were identified from our database. Hospitalised patients with Campylobacter spp. were then matched for age and gender with patients hospitalised for gastroenteritis of other or unknown aetiology. Patients who had undergone abdominal radiographic investigation or had received a surgery consultation were included as “acute abdomen” (AA) cases. Demographics, clinical characteristics and management were compared between AA and non-AA cases. RESULTS: One hundred and forty-one patients with cultures positive for Campylobacter spp. were included in the analysis. Nineteen patients were identified as AA cases. Fewer of these had diarrhoea (14/19, 74% vs 117/121, 97%; p = 0.02) and more reported a lower sense of general wellbeing (8/18, 44% vs 8/108, 7%; p

The identification of microorganisms directly from blood cultures using MALDI-TOF MS has been shown to be the most impacting application of this methodology. In this study, a novel commercial method was evaluated in four clinical microbiology laboratories. Positive blood culture samples (n = 801) were processed using a rapid BACpro® II kit and then compared with the routine gold standard. A subset of monomicrobial BCs (n = 560) were analyzed in parallel with a Sepsityper® Kit (Bruker Daltonics, Bremen, Germany) and compared with the rapid BACpro® II kit. In addition, this kit was also compared with two different in-house methods. Overall, 80.0% of the monomicrobial isolates (609/761; 95% CI 71.5–88.5) were correctly identified by the rapid BACpro® II kit at the species level (92.3% of the Gram negative and 72.4% of the Gram positive bacteria). The comparison with the Sepsityper® Kit showed that the rapid BACpro® II kit generated higher rates of correct species-level identification for all categories (p > 0.0001), except for yeasts identified with score values > 1.7. It also proved superior to the ammonium chloride method (p > 0.0001), but the differential centrifugation method allowed for higher rates of correct identification for Gram negative bacteria (p > 0.1). The percentage of accurate species-level identification of Gram positive bacteria was particularly noteworthy in comparison with other commercial and in-house methods.

Among the species Mycobacterium kansasii, seven subtypes have been previously reported based on the PCR and the restriction fragment length polymorphism of the gene hsp65. Here, we used whole-genome sequencing to refine M. kansasii taxonomy and correct multiple inconsistencies. Average nucleotide identity (ANI) values between M. kansasii subtypes ranged from 88.4 to 94.2 %, lower than the accepted 95-96 % cut-off for species delineation. In addition, Mycobacterium gastri was closer to the M. kansasii subtypes 1, 2, 3, 4 and 5 than M. kansasii subtype 6. The recently described species Mycobacterium persicum shared 99.77 % ANI with M. kansasii subtype 2. Consistent with the ANI results, the digital DNA-DNA hybridization value was below the 70 % threshold for species delineation between subtypes and above it within subtypes as well as between subtype 2 and M. persicum. Furthermore, core-genome phylogeny confirmed the current M. kansasii species to be polyphyletic. Hence, we propose (i) Mycobacterium pseudokansasii sp. nov., replacing subtype 3, with the type strain MK142T(=CCUG 72128T=DSM 107152T), (ii) Mycobacterium innocens sp. nov., replacing subtype 5, with the type strain MK13T (=CCUG 72126T=DSM 107161T), and (iii) Mycobacterium attenuatum sp. nov., replacing subtype 6, with the type strain MK41T(=CCUG 72127T=DSM 107153T). Subtype 4 represents a new species-level lineage based on the genomic data but no strain was available. No genome sequence or strain was available for subtype 7. The proposed nomenclature will facilitate the identification of the most pathogenic subtype 1 as M. kansasii by clinicians while the new species names suggest the attenuated pathogenicity of the other subtypes.

In clinical practice, fecal microbiota transplantation (FMT) has been established as an unparalleled therapy to date for multiple recurrent Clostridioides difficile infections (CDI). The implementation of the FMT in practice requires a significant investment to meet legal, security and financial requirements. Research on the microbiota is booming and multiple investigations on FMT in indications other than CDI are ongoing.En pratique clinique, la transplantation de microbiote fécal (TMF) s’est établie comme une thérapie sans équivalent à ce jour pour les infections à

ObjectivesIdentification of microorganisms directly from blood cultures (BCs) using MALDI-TOF MS has shown to be the application with most impact in this methodology. In this study, a novel commercial method, the rapidBACpro® II, was evaluated in four clinical microbiology laboratories.MethodsPositive blood culture samples (n=801) were processed using the rapidBACpro® II kit and then compared with routine gold standard. A subset of monomicrobial BCs (n=560) were analyzed in parallel with the Sepsityper® kit (Bruker Daltonics, Bremen, Germany) and compared with the rapidBACpro® II kit. In addition, the rapidBACpro® II kit was also compared with two different in-house methods.ResultsOverall, 80.0% of the monomicrobial isolates (609/761) were correctly identified by the rapidBACpro® II kit at the species level (92.3% of the Gram negative and 72.4% of the Gram positive bacteria). The comparison with the Sepsityper® kit yielded higher rates of correct species-level identification provided by the rapidBACpro® II kit for all categories (p>0.0001) except for yeasts identified with score values >1.7. It also proved superior to the ammonium chloride method (p>0.0001) but the differential centrifugation method allowed higher rates of correct identification for Gram negative bacteria (p>0.1).ConclusionsThe rapidBACpro® II kit allowed a high rate of microorganisms correctly identified. The percentage of accurate species-level identification of Gram positive bacteria was particularly noteworthy in comparison with other commercial and in-house methods. This fact was especially interesting in the case of Staphylococcus sp. and Streptococcus sp. in order to elucidate their clinical impact, for example in device-associated bacteremia.

Clostridioides difficile is an increasingly common pathogen both within and outside the hospital and is responsible for a large clinical spectrum from asymptomatic carriage to complicated infection associated with a high mortality. While diagnostic methods have considerably progressed over the years, the optimal diagnostic algorithm is still debated and there is no single diagnostic test that can be used as a standalone test. More importantly, the heterogeneity in diagnostic practices between centers along with the lack of robust surveillance systems in all countries and an important degree of underdiagnosis due to lack of clinical suspicion in the community, hinder a more accurate evaluation of the burden of disease. Our improved understanding of the physiopathology of CDI has allowed some significant progress in the treatment of CDI, including a broader use of fidaxomicine, the use of fecal microbiota transplantation for multiples recurrences and newer approaches including antibodies, vaccines and new molecules, already developed or in the pipeline. However, the management of CDI recurrences and severe infections remain challenging and the main question remains: how to best target these often expensive treatments to the right population. In this review we discuss current diagnostic approaches, treatment and potential prevention strategies, with a special focus on recent advances in the field as well as areas of uncertainty and unmet needs and how to address them.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a common healthcare-associated pathogen that remains a major public health concern. Sequence type 228 (ST228) was first described in Germany and spread to become a successful MRSA clone in several European countries. In 2000, ST228 emerged in Lausanne and has subsequently caused several large outbreaks. Here, we describe the evolutionary history of this clone and identify the genetic changes underlying its expansion in Switzerland. Materials and Methods: We aimed to understand the phylogeographic and demographic dynamics of MRSA ST228/ST111 by sequencing 530 representative isolates of this clone that were collected from 14 European countries between 1997 and 2012. Results: The phylogenetic analysis revealed distinct lineages of ST228 isolates associated with specific geographic origins. In contrast, isolates of ST111, which is a single locus variant of ST228 sharing the same spa type t041, formed a monophyletic cluster associated with multiple countries. The evidence points to a German origin of the sampled population, with the basal German lineage being characterized by spa type t001. The highly successful Swiss ST228 lineage diverged from this progenitor clone through the loss of the aminoglycoside-streptothricin resistance gene cluster and the gain of mupirocin resistance. This lineage was introduced first in Geneva and was subsequently introduced into Lausanne. Conclusion: Our results reveal the radiation of distinct lineages of MRSA ST228 from a German progenitor, as the clone spread into different European countries. In Switzerland, ST228 was introduced first in Geneva and was subsequently introduced into Lausanne., SCOPUS: ar.j, info:eu-repo/semantics/published

Objectives Becton-Dickinson recently developed the Phoenix™ CPO (carbapenemase-producing organism) Detect Test, a growth-based test embedded in Gram-negative (GN) panels for the detection and confirmation of bacteria producing class A, B and D carbapenemases. This study aimed to (a) determine the performance of the CPO test, and (b) assess its added value in routine diagnostic workflows. Methods The performance of the BD Phoenix CPO test was analysed retrospectively on a collection of 185 molecularly characterized strains, including 92 CPOs, and prospectively on 135 and 160 routine isolates with and without CPO suspicion, respectively. Results In the retrospective study the CPO test exhibited 92.4% accuracy (95%CI 87.6–95.8), 97.8% sensitivity (95%CI 92.4–99.7) and 87.1% specificity (95%CI 78.6–93.2) for carbapenemase detection. The CPO test provided a classification to class A, B, and D for 81.3% of detected carbapenemases with 94.6% accuracy (95%CI 86.7–98.5). In the prospective study the CPO test detection performance showed 77.8% accuracy (95%CI 68.8–84.5), 100% sensitivity (95%CI 91.2–100) and 67.8% specificity (95%CI 57.3–77.1) with 135 CPO-suspicious isolates and 98.8% accuracy and specificity (95%CI 95.6–99.9) with 160 non-CPO-suspicious isolates. Compared to routine testing, the implementation of the CPO test allowed a mean reduction of 21.3 h (95%CI 17.6–25) in turnaround time, 16.8 min (95%CI 13.4–20.2) in hands-on time, and 20.6 CHF (95%CI 16.5–24.8) in costs. Conclusions The CPO test is reliable for the detection of CPO with a high sensitivity. However, the relatively low detection specificity required the use of additional confirmatory methods. The carbapenemase classification accuracy is robust in providing preliminary results before molecular characterization. Finally, the implementation of the test in routine workflows allowed a significant reduction in turnaround time, hands-on time and cost compared to the conventional approach.

Pseudomonas aeruginosa is one of the main pathogens responsible for nosocomial infections, particularly in Intensive Care Units (ICUs). Due to the complexity of P. aeruginosa ecology, only powerful typing methods can efficiently allow its surveillance and the detection during expanding outbreaks. An increase in P. aeruginosa incidence was observed in the ICUs of the Lausanne University Hospital between 2010 and 2014. All clinical and environmental isolates retrieved during this period were typed with Double locus sequence typing (DLST), which detected the presence of three major genotypes: DLST 1–18, DLST 1–21, and DLST 6–7. DLST 1–18 (ST1076) isolates were previously associated with an epidemiologically well-described outbreak in the burn unit. Nevertheless, DLST 1–21 (ST253) and DLST 6–7 (ST17) showed sporadic occurrence with only few cases of possible transmission between patients. Whole genome sequencing (WGS) was used to further investigate the epidemiology of these three major P. aeruginosa genotypes in the ICUs. WGS was able to differentiate between outbreak and non-outbreak isolates and confirm suspected epidemiological links. Additionally, whole-genome single nucleotide polymorphisms (SNPs) results considered isolates as closely related for which no epidemiological links were suspected, expanding the epidemiological investigation to unsuspected links. The combination of a first-line molecular typing tool (DLST) with a more discriminatory method (WGS) proved to be an accurate and cost-efficient typing strategy for the investigation of P. aeruginosa epidemiology in the ICUs.

Candida albicans causes life-threatening systemic infections in immunosuppressed patients. These infections are commonly treated with fluconazole, an antifungal agent targeting the ergosterol biosynthesis pathway. Current Antifungal Susceptibility Testing (AFST) methods are time-consuming and are often subjective. Moreover, they cannot reliably detect the tolerance phenomenon, a breeding ground for the resistance. An alternative to the classical AFST methods could use Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry (MS). This tool, already used in clinical microbiology for microbial species identification, has already offered promising results to detect antifungal resistance on non-azole tolerant yeasts. Here, we propose a machine-learning approach, adapted to MALDI-TOF MS data, to qualitatively detect fluconazole resistance in the azole tolerant species C. albicans. MALDI-TOF MS spectra were acquired from 33 C. albicans clinical strains isolated from 15 patients. Those strains were exposed for 3 h to 3 fluconazole concentrations (256, 16, 0 μg/mL) and with (5 μg/mL) or without cyclosporin A, an azole tolerance inhibitor, leading to six different experimental conditions. We then optimized a protein extraction protocol allowing the acquisition of high-quality spectra, which were further filtered through two quality controls. The first one consisted of discarding not identified spectra and the second one selected only the most similar spectra among replicates. Quality-controlled spectra were divided into six sets, following the sample preparation's protocols. Each set was then processed through an R based script using pre-defined housekeeping peaks allowing peak spectra positioning. Finally, 32 machine-learning algorithms applied on the six sets of spectra were compared, leading to 192 different pipelines of analysis. We selected the most robust pipeline with the best accuracy. This LDA model applied to the samples prepared in presence of tolerance inhibitor but in absence of fluconazole reached a specificity of 88.89% and a sensitivity of 83.33%, leading to an overall accuracy of 85.71%. Overall, this work demonstrated that combining MALDI-TOF MS and machine-learning could represent an innovative mycology diagnostic tool.

Whole-genome sequencing (WGS) has emerged as a powerful molecular typing method for outbreak analysis enabling the rapid discrimination between outbreak and non-outbreak isolates. However, such analysis can be challenging in the absence of closely related reference genomes. In this study, we assessed the use of WGS in investigating an outbreak of a relatively understudied bacterial pathogen with no publicly available closely related reference genome. Eleven Burkholderia cepacia complex (Bcc) isolates (seven from patients and four from disposable dermal gloves packages) that were collected during an outbreak were sequenced using the Illumina MiSeq platform. Our results showed that mapping the 11 sequenced Bcc outbreak isolates against a genetically distant reference genome yield loses coverage (31.6-48.3%) and a high number of detected false single-nucleotide polymorphisms (SNPs) (1123-2139). Therefore, a reference genome consensus from an outbreak clinical isolate was generated by combining both de novo assembly and mapping approaches. Based on this approach, we were able to demonstrate that the Bcc outbreak isolates were closely related and were phylogenetically distinct from the 11 publically available Bcc genomes. In addition, the pairwise SNP distance analysis detected only 1 to 6 SNPs differences among the outbreak isolates, confirming that contaminated disposable dermal gloves were the cause of the outbreak.

Atopic dermatitis (AD) has a well-established association with skin colonization or infection by Staphylococcus aureus, which can exacerbate the disease. However, a causal relationship between specific changes in skin colonization during the first years of life and AD development still remains unclear. In this prospective birth cohort study, we aimed to characterize the association between skin colonization and AD development in 149 white infants with or without a family history of atopy. We assessed infants clinically and collected axillary and antecubital fossa skin swabs for culture-based analysis at birth and at seven time points over the first 2 years of life. We found that at age 3 months, S. aureus was more prevalent on the skin of infants who developed AD later on. S. aureus prevalence was increased on infants' skin at the time of AD onset and also 2 months before it, when compared with age-matched, unaffected infants. Furthermore, at AD onset, infants testing positive for S. aureus were younger than uncolonized subjects. In conclusion, our results suggest that specific changes in early-life skin colonization may actively contribute to clinical AD onset in infancy.

Bacillus cereus bacteraemia can be severe, especially among patients with haematologic malignancy. We retrospectively reviewed first episodes of true B. cereus bacteraemia (more than one positive bottle plus signs of infection) at our institution between 1997 and 2013 with the aim to compare haematologic versus nonhaematologic patients and analyse episodes with complicated outcome. Among 56 episodes of positive-blood cultures for B. cereus, 21 were considered significant. Median age was 54 years (range 23–82 years). Ten patients (48%) had a haematologic malignancy; all were neutropenic at the time of B. cereus bacteraemia. Nonhaematologic patients were either intravenous drug users (n = 3, 14%), polytraumatized (n = 3, 14%) or had multiple chronic comorbidities (n = 5, 24%). Most episodes were hospital acquired (15, 71%). Sources of bacteraemia were intravascular catheter (n = 11, 52%), digestive tract (n = 6, 29%), drug injection (n = 3, 14%) and wound (n = 1, 5%). Adequate antibiotic therapy was provided to 18 patients (86%) during a median of 17 days (range 2–253 days). The intravascular catheter was removed in eight cases (42%). Three haematologic patients had a complicated course with neurologic complications (meningoencephalitis and cerebral abscesses). Complications appeared to be associated with catheter infection (100% of complicated cases vs. 29% of noncomplicated cases). In conclusion, B. cereus bacteraemia can have a complicated course in a subset of patients, mainly those with haematologic malignancy. Catheter infection may be associated with a worse outcome with frequent neurologic complications.

A novel KPC variant, KPC-41, was identified in a Klebsiella pneumoniae clinical isolate from Switzerland. This β-lactamase possessed a 3-amino-acid insertion (Pro-Asn-Lys) located between amino acids 269 and 270 compared to the KPC-3 amino acid sequence. Cloning and expression of the bla(KPC-41) gene in Escherichia coli, followed by determination of MIC values and kinetic parameters, showed that KPC-41, compared to those of KPC-3, has an increased affinity to ceftazidime and a decreased sensitivity to avibactam, leading to resistance to ceftazidime-avibactam once produced in K. pneumoniae. Furthermore, KPC-41 exhibited a drastic decrease of its carbapenemase activity. This report highlights that a diversity of KPC variants conferring resistance to ceftazidime-avibactam already circulate in Europe.

Candida albicans causes life-threatening systemic infections in immunosuppressed patients. These infections are commonly treated with fluconazole, an antifungal agent targeting the ergosterol biosynthesis pathway. Current Antifungal Susceptibility Testing (AFST) methods are time-consuming and are often subjective. Moreover, they cannot reliably detect the tolerance phenomenon, a breeding ground for the resistance. An alternative to the classical AFST methods could use Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry (MS). This tool, already used in clinical microbiology for microbial species identification, has already offered promising results to detect antifungal resistance on non-azole tolerant yeasts. Here, we propose a machine-learning approach, adapted to MALDI-TOF MS data, to qualitatively detect fluconazole resistance in the azole tolerant species C. albicans. MALDI-TOF MS spectra were acquired from 33 C. albicans clinical strains isolated from 15 patients. Those strains were exposed for 3 h to 3 fluconazole concentrations (256, 16, 0 μg/mL) and with (5 μg/mL) or without cyclosporin A, an azole tolerance inhibitor, leading to six different experimental conditions. We then optimized a protein extraction protocol allowing the acquisition of high-quality spectra, which were further filtered through two quality controls. The first one consisted of discarding not identified spectra and the second one selected only the most similar spectra among replicates. Quality-controlled spectra were divided into six sets, following the sample preparation’s protocols. Each set was then processed through an R based script using pre-defined housekeeping peaks allowing peak spectra positioning. Finally, 32 machine-learning algorithms applied on the six sets of spectra were compared, leading to 192 different pipelines of analysis. We selected the most robust pipeline with the best accuracy. This LDA model applied to the samples prepared in presence of tolerance inhibitor but in absence of fluconazole reached a specificity of 88.89% and a sensitivity of 83.33%, leading to an overall accuracy of 85.71%. Overall, this work demonstrated that combining MALDI-TOF MS and machine-learning could represent an innovative mycology diagnostic tool.

En raison d’un portage asymptomatique significatif, le diagnostic d’infection a Clostridioides difficile reste un defi diagnostique.

The visual analysis of peripheral blood samples is an important test for blood illnesses diagnosis, like leukaemia or malaria. Malaria is an epidemic health disease and a rapid, accurate diagnosis is necessary for proper intervention. Generally, pathologists visually examine blood stained slides for malaria diagnosis. Nevertheless, this kind of visual inspection is subjective, error-prone and time-consuming. In order to overcome these issues, numerous methods of automatic malaria diagnosis have been proposed so far. Unfortunately, no public image dataset is available to test and compare such algorithms. The aim of this paper is to present the first public dataset of blood samples afflicted by malaria, specifically designed to evaluate and compare algorithms for segmentation and classification of malaria parasite species. Every image is provided with its related ground truth and parasite’s classification of type and stage of life. Our purpose is to offer a new comparative test tool to the image processing and pattern matching communities, in order to encourage and improve computer-aided malaria parasites analysis.

Objectives To assess recent trends in susceptibility to antibiotics among urinary isolates isolated in European emergency departments (EDs) and to identify isolates with a high (90% or more) predicted probability of susceptibility to fluoroquinolones or third-generation cephalosporins (3GCs). Methods In this cross-sectional study, we included urine cultures obtained from adult patients between 2010 and 2016 in 24 European EDs. Temporal trends were assessed using time-series analysis and multivariate logistic models. Multivariate logistic models were also used to predict susceptibility to fluoroquinolones or 3GCs from patient age and sex, year, month and ED. Results We included 88242 isolates. Time-series analysis found a significant increase in susceptibility to fluoroquinolones and no significant trend for susceptibility to 3GCs. Adjusting for patient age and sex, ED and organism, multivariate models showed that susceptibility to 3GCs decreased from 2014 to 2016, while susceptibility to fluoroquinolones increased in 2015 and 2016. Among isolates from 2016, multivariate models predicted high probability of susceptibility to fluoroquinolones in 11% of isolates (positive predictive value 91%) and a high probability of susceptibility to 3GCs in 35% of isolates (positive predictive value 94%). Conclusions Susceptibility of ED urinary isolates to fluoroquinolones increased from 2014, while susceptibility to 3GCs decreased from 2015. Predictive models identified isolates with a high probability of susceptibility to fluoroquinolones or 3GCs. The ability of such models to guide the empirical treatment of pyelonephritis in the ED remains to be determined.

Resume Les laboratoires de diagnostic moleculaire et de serologie ont ete automatises depuis de nombreuses annees. Aujourd’hui, la bacteriologie entre egalement dans l’ere de l’automatisation grâce a de nouvelles solutions IT, grâce a l’usage de milieux liquides pour les frottis, et grâce a la disponibilite du MALDI-TOF pour l’identification bacterienne. Cette automatisation des laboratoires diagnostiques ameliore l’efficience, reduit les couts et peut aussi reduire a terme le temps de rendu des resultats par une detection precoce de la croissance des colonies a l’aide de nouveaux algorithmes de detection de leur croissance. Cependant, pour tirer pleinement parti des avantages des solutions automatisees, il ne faut pas envisager une simple robotisation des activites humaines, mais reorganiser de maniere globale les procedures et le workflow. Ainsi, l’automatisation pourra reduire les delais de rendu de resultats et ameliorer significativement la qualite avec un benefice escompte sur les soins aux patients.

BACKGROUND: Empirical antibacterial therapy in hospitals is usually guided by local epidemiologic features reflected by institutional cumulative antibiograms. We investigated additional information inferred by aggregating cumulative antibiograms by type of unit or according to the place of acquisition (i.e. community vs. hospital) of the bacteria. MATERIALS AND METHODS: Antimicrobial susceptibility rates of selected pathogens were collected over a 4-year period in an university-affiliated hospital. Hospital-wide antibiograms were compared with those selected by type of unit and sampling time (48 h after hospital admission). RESULTS: Strains isolated >48 h after admission were less susceptible than those presumably arising from the community (48 h after admission. When compared to hospital-wide antibiograms, susceptibility rates were lower in the ICU and surgical units for Escherichia coli to amoxicillin-clavulanate, enterococci to penicillin, and Pseudomonas aeruginosa to anti-pseudomonal beta-lactams, and in medical units for Staphylococcus aureus to oxacillin. In contrast, few differences were observed among strains isolated within 48 h of admission. CONCLUSIONS: Hospital-wide antibiograms reflect the susceptibility pattern for a specific unit with respect to community-acquired, but not to hospital-acquired strains. Antibiograms adjusted to these parameters may be useful in guiding the choice of empirical antibacterial therapy.

The quality of sample inoculation is critical for achieving an optimal yield of discrete colonies in both monomicrobial and polymicrobial samples to perform identification and antibiotic susceptibility testing. Consequently, we compared the performance between the InoqulA (BD Kiestra), the WASP (Copan), and manual inoculation methods. Defined mono- and polymicrobial samples of 4 bacterial species and cloudy urine specimens were inoculated on chromogenic agar by the InoqulA, the WASP, and manual methods. Images taken with ImagA (BD Kiestra) were analyzed with the VisionLab version 3.43 image analysis software to assess the quality of growth and to prevent subjective interpretation of the data. A 3- to 10-fold higher yield of discrete colonies was observed following automated inoculation with both the InoqulA and WASP systems than that with manual inoculation. The difference in performance between automated and manual inoculation was mainly observed at concentrations of >10 6 bacteria/ml. Inoculation with the InoqulA system allowed us to obtain significantly more discrete colonies than the WASP system at concentrations of >10 7 bacteria/ml. However, the level of difference observed was bacterial species dependent. Discrete colonies of bacteria present in 100- to 1,000-fold lower concentrations than the most concentrated populations in defined polymicrobial samples were not reproducibly recovered, even with the automated systems. The analysis of cloudy urine specimens showed that InoqulA inoculation provided a statistically significantly higher number of discrete colonies than that with WASP and manual inoculation. Consequently, the automated InoqulA inoculation greatly decreased the requirement for bacterial subculture and thus resulted in a significant reduction in the time to results, laboratory workload, and laboratory costs.

Blood culture remains the best approach to identify the incriminating microorganisms when a bloodstream infection is suspected, and to guarantee that the antimicrobial treatment is adequate. Major improvements have been made in the last years to increase the sensitivity and specificity and to reduce the time to identification of microorganisms recovered from blood cultures. Among other factors, the introduction in clinical microbiology laboratories of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology revolutionized the identification of microorganisms whereas the introduction of nucleic-acid-based methods, such as DNA hybridization or rapid PCR-based test, significantly reduce the time to results. Together with traditional antimicrobial susceptibility testing, new rapid methods for the detection of resistance mechanisms respond to major epidemiological concerns such as methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase or carbapenemases. This review presents and discusses the recent developments in microbial diagnosis of bloodstream infections based on blood cultures.

Background Automation in microbiology laboratories impacts management, workflow, productivity and quality. Further improvements will be driven by the development of intelligent image analysis allowing automated detection of microbial growth, release of sterile samples, identification and quantification of bacterial colonies and reading of AST disk diffusion assays. We investigated the potential benefit of intelligent imaging analysis by developing algorithms allowing automated detection, semi-quantification and identification of bacterial colonies. Methods Defined monomicrobial and clinical urine samples were inoculated by the BD Kiestra™ InoqulA™ BT module. Image acquisition of plates was performed with the BD Kiestra™ ImagA BT digital imaging module using the BD Kiestra™ Optis™ imaging software. The algorithms were developed and trained using defined data sets and their performance evaluated on both defined and clinical samples. Results The detection algorithms exhibited 97.1% sensitivity and 93.6% specificity for microbial growth detection. Moreover, quantification accuracy of 80.2% and of 98.6% when accepting a 1 log tolerance was obtained with both defined monomicrobial and clinical urine samples, despite the presence of multiple species in the clinical samples. Automated identification accuracy of microbial colonies growing on chromogenic agar from defined isolates or clinical urine samples ranged from 98.3% to 99.7%, depending on the bacterial species tested. Conclusion The development of intelligent algorithm represents a major innovation that has the potential to significantly increase laboratory quality and productivity while reducing turn-around-times. Further development and validation with larger numbers of defined and clinical samples should be performed before transferring intelligent imaging analysis into diagnostic laboratories.

Kingella kingae, producing the cytotoxic RTX protein, is a causative agent of serious infections in humans such as bacteremia, endocarditis and osteoarticular infection, especially in young children. Recently, Kingella negevensis, a related species, has been isolated from the oral cavity of healthy children. In this study, we report the isolation of K. negevensis strain eburonensis, initially misidentified as K. kingae with MALDI-TOF MS, from a vaginal specimen of a patient suffering of vaginosis. The genome sequencing and analysis of this strain together with comparative genomics of the Kingella genus revealed that K. negevensis possesses a full homolog of the rtx operon of K. kingae involved in the synthesis of the RTX toxin. We report that a K. kingae specific diagnostic PCR, based on the rtxA gene, was positive when tested on K. negevensis strain eburonensis DNA. This cross-amplification, and risk of misidentification, was confirmed by in silico analysis of the target gene sequence. To overcome this major diagnostic issue we developed a duplex real-time PCR to detect and distinguish K. kingae and K. negevensis. In addition to this, the identification of K. negevensis raises a clinical issue in term of pathogenic potential given the production of a RTX hemolysin.

BACKGROUND: Early identification of pathogens from blood cultures using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry may optimize the choice of empirical antibiotic therapy in the setting of bloodstream infections. We aimed to assess the impact of this new technology on the use of antibiotic treatment in patients with gram-negative bacteremia. METHODS: We conducted a prospective observational study from January to December 2010 to evaluate the sequential and separate impacts of Gram stain reporting and MALDI-TOF bacterial identification performed on blood culture pellets in patients with gram-negative bacteremia. The primary outcome was the impact of MALDI-TOF on empirical antibiotic choice. RESULTS: Among 202 episodes of gram-negative bacteremia, Gram stain reporting had an impact in 42 cases (20.8%). MALDI-TOF identification led to a modification of empirical therapy in 71 of all 202 cases (35.1%), and in 16 of 27 cases (59.3%) of monomicrobial bacteremia caused by AmpC-producing Enterobacteriaceae. The most frequently observed impact was an early appropriate broadening of the antibiotic spectrum in 31 of 71 cases (43.7%). In total, 143 of 165 episodes (86.7%) of monomicrobial bacteremia were correctly identified at genus level by MALDI-TOF. CONCLUSIONS: In a low prevalence area for extended spectrum betalactamases (ESBL) and multiresistant gram-negative bacteria, MALDI-TOF performed on blood culture pellets had an impact on the clinical management of 35.1% of all gram-negative bacteremia cases, demonstrating a greater impact than Gram stain reporting. Thus, MALDI-TOF could become a vital second step beside Gram stain in guiding the empirical treatment of patients with bloodstream infection.

We report here the draft genome sequences of two multidrug-resistant Acinetobacter baumannii clinical strains, H31499 and H31506, which were isolated at the Lausanne University Hospital in 2015 from an Albanian and a Togolese patient, respectively.

We report here the draft genome sequences of two multidrug-resistant Acinetobacter baumannii clinical strains, H31499 and H31506, which were isolated at the Lausanne University Hospital in 2015 from an Albanian and a Togolese patient, respectively.