From Bed to Bench: Pre-analytical Stability of 29 Anti-infective Agents in Plasma and Whole Blood to Improve Accuracy of Therapeutic Drug Monitoring.

BACKGROUND: Therapeutic drug monitoring requires a validated assay and appropriate conditions for sample shipment and storage based on the stability of the compound to be analyzed. This study evaluated the stability of 29 antimicrobial compounds in whole blood (WB) and plasma samples under various storage conditions. METHODS: The pre-analytical stability of 22 antibiotics (amoxicillin, aztreonam, cefazolin, cefepime, cefotaxime, cefoxitin, ceftazidime, ceftobiprole, ceftolozane, ceftriaxone, ciprofloxacin, clindamycin, cloxacillin, daptomycin, levofloxacin, linezolid, meropenem, metronidazole, moxifloxacin, piperacillin, sulfamethoxazole, and trimethoprim), 2 beta-lactamase inhibitors (avibactam, tazobactam), and 5 antituberculosis drugs (ethambutol, isoniazid, pyrazinamide, rifabutin, and rifampicin) was assessed by WB for up to 24 hours at room temperature (RT) and 72 hours at +4°C. The stability in plasma was evaluated for up to 6 hours at RT, 24 hours at +4°C, 1 month at -20°C, and 6 months at -80°C. RESULTS: Concerning WB stability, all investigated compounds were stable for 24 hours at RT, except meropenem and isoniazid, which were stable for 6 hours; however, for 24 hours at +4°C, all the compounds were stable. For storage durations of 48 and 72 hours at +4°C, all compounds were stable, except for ciprofloxacin, cotrimoxazole, and isoniazid. Concerning stability in plasma, all compounds were stable for 6 hours at RT, and all except isoniazid were stable for 24 hours at +4°C. All the tested compounds were stable for 7 days at -20°C, except isoniazid, for which a degradation of approximately 20% was observed. An important degradation was observed for beta-lactam antibiotics after 1 month at -20°C. All compounds were stable at -80°C for 6 months. CONCLUSIONS: The pre-analytical stabilities of several anti-infective compounds was described. The present results can be used to determine the appropriate conditions for shipping and storing samples dedicated to therapeutic drug monitoring of the investigated compounds.

Risk factors for Pseudomonas aeruginosa VIM colonization/infection in the ICU: Case control study.

BACKGROUND: Carbapenem-resistant strains of Pseudomonas aeruginosa (CRPA) have become a major healthcare concern in many countries, against which anti-infective strategies are limited and which require adequate infection control interventions. Knowing the different modes of transmission of CRPA in intensive care units (ICUs) would be helpful to adapt the means of prevention. METHODS: The aim of this retrospective case-control study was conducted between 01/01/2017 and 02/28/2022 to identify the risk factors for the acquisition of CRPA in ICUs. RESULTS: During the study period, 147 patients were included (49 cases and 98 controls). Among the 49 patients, 31 (63%) acquired CRPA in clusters and 18 (37%) sporadically. An univariate analysis showed that five variables were associated with CRPA acquisition including (i) prior antibiotic prescriptions, (ii) admission to rooms 203 and 207, (iii) severity of illness at admission, and (iv) use of mechanical ventilation. Multivariate analysis identified three factors of CRPA acquisition including admission to room 203 (OR = 29.5 [3.52-247.09]), previous antibiotic therapy (OR = 3.44 [1.02 - 11.76]) and severity of condition at admission (OR = 1.02 [1 - 1.04]). CONCLUSION: Our study suggests the role of a contaminated environment in the acquisition of CRPA in the ICU, along with antibiotic use.

Bloodstream Infections: Comparison of Diagnostic Methods and Therapeutic Consequences between a Hospital in a Resource-Limited Setting and Two French Hospitals.

In recent years, the diagnosis of bloodstream infections has been complemented by rapid microbiological methods, unattainable to most clinical laboratories in resource-limited settings. We evaluated the impact of their shortage on antibiotic therapy adequacy. We conducted a prospective multicenter cohort study including 150 adult Gram-negative bacilli bacteremia episodes, evenly distributed across three university hospitals: one in Lebanon, a resource-limited setting, and two in France, a resource-rich setting. Previous colonization by multidrug-resistant organisms (MDRO) was significantly more prevalent among the Lebanese than the French group of patients (16/50 vs. 5/100; p < 0.01). Bloodstream infections by carbapenemase-producing Enterobacterales and other MDRO were higher among the Lebanese than the French group of patients (25/50 vs. 12/100; p < 0.01). For the French group, rapid identification of species and mechanisms of resistance significantly shortened turnaround time for definitive laboratory diagnosis and increased antibiotic therapy adequacy. No statistically significant differences were noted in targeted antibiotic therapy between the two groups. This study suggests that, in settings where bacterial resistance is prevalent, rapid microbiological methods have not provided any additional value. The clinical and economic impact of rapid microbiological methods will likely depend on local CPE, VRE, and other MDRO epidemiology and are areas for future research.

The bacterial genetic determinants of Escherichia coli capacity to cause bloodstream infections in humans.

Escherichia coli is both a highly prevalent commensal and a major opportunistic pathogen causing bloodstream infections (BSI). A systematic analysis characterizing the genomic determinants of extra-intestinal pathogenic vs. commensal isolates in human populations, which could inform mechanisms of pathogenesis, diagnostic, prevention and treatment is still lacking. We used a collection of 912 BSI and 370 commensal E. coli isolates collected in France over a 17-year period (2000-2017). We compared their pangenomes, genetic backgrounds (phylogroups, STs, O groups), presence of virulence-associated genes (VAGs) and antimicrobial resistance genes, finding significant differences in all comparisons between commensal and BSI isolates. A machine learning linear model trained on all the genetic variants derived from the pangenome and controlling for population structure reveals similar differences in VAGs, discovers new variants associated with pathogenicity (capacity to cause BSI), and accurately classifies BSI vs. commensal strains. Pathogenicity is a highly heritable trait, with up to 69% of the variance explained by bacterial genetic variants. Lastly, complementing our commensal collection with an older collection from 1980, we predict that pathogenicity continuously increased through 1980, 2000, to 2010. Together our findings imply that E. coli exhibit substantial genetic variation contributing to the transition between commensalism and pathogenicity and that this species evolved towards higher pathogenicity.

What to Do with the New Antibiotics?

Multidrug-resistant Gram-negative bacteria-related infections have become a real public health problem and have exposed the risk of a therapeutic impasse. In recent years, many new antibiotics have been introduced to enrich the therapeutic armamentarium. Among these new molecules, some are mainly of interest for the treatment of the multidrug-resistant infections associated with Pseudomonas aeruginosa (ceftolozane/tazobactam and imipenem/relebactam); others are for carbapenem-resistant infections associated with Enterobacterales (ceftazidime/avibactam, meropenem/vaborbactam); and finally, there are others that are effective on the majority of multidrug-resistant Gram-negative bacilli (cefiderocol). Most international guidelines recommend these new antibiotics in the treatment of microbiologically documented infections. However, given the significant morbidity and mortality of these infections, particularly in the case of inadequate therapy, it is important to consider the place of these antibiotics in probabilistic treatment. Knowledge of the risk factors for multidrug-resistant Gram-negative bacilli (local ecology, prior colonization, failure of prior antibiotic therapy, and source of infection) seems necessary in order to optimize antibiotic prescriptions. In this review, we will assess these different antibiotics according to the epidemiological data.

Using gamification to improve engagement and learning outcomes in medical microbiology: the case study of 'BacteriaGame'.

The fight against antibiotic resistance has become a true global public health challenge of gargantuan proportions. Amongst the myriad of approaches being explored to tackle this predicament, one strategy involves enhancing prescriber knowledge and in particular their basic knowledge of medical bacteriology. Yet, as we well know in medical microbiology teachings, traditional lectures can be arduous, attempting to cram in a vast array of information in a limited time. An alternative solution to improve student engagement and enhance learning outcomes is to utilize educational games in complementary approach. Such games are an effective means of inspiring students to learn, encouraging self-assessment, and injecting diversity into the teaching process. To this end, we have developed and evaluated an educational card game, the 'BacteriaGame,' aimed at our medical students in medical bacteriology. Designed for students at the basic level, it serves as activity at the end of their apprenticeship to their bacteriology education. Additionally, it can also be used as a review tool by more advanced students, with teachers able to impart additional knowledge as the game progresses. We also use it in continuous training of medical laboratory staff. In this study, we evaluated the game at various stages of medical education, collecting feedback and analysing its impact on knowledge acquisition, comparing it to traditional lectures. Feedback from the majority of students revealed that the rules were clear, the game was enjoyable, and neither too lengthy nor too challenging. The integration of 'BacteriaGame' into their future training piqued their interest. In terms of learning outcomes, we discovered a significant increase in knowledge acquisition among those who used the game (P < .05). 'BacteriaGame' is now published by the French Society of Microbiology (SFM) and distributed in all medical and pharmacy schools thanks to a funding of the French Health Ministry. An English edition of the game is also available for international use as a physical copy to be purchased from the SFM. This will allow a large-scale distribution to colleagues who would like to use this game in their teaching.

When and How to Use MIC in Clinical Practice?

Bacterial resistance to antibiotics continues to be a global public health problem. The choice of the most effective antibiotic and the use of an adapted dose in the initial phase of the infection are essential to limit the emergence of resistance. This will depend on (i) the isolated bacteria and its resistance profile, (ii) the pharmacodynamic (PD) profile of the antibiotic used and its level of toxicity, (iii) the site of infection, and (iv) the pharmacokinetic (PK) profile of the patient. In order to take account of both parameters to optimize the administered treatment, a minimal inhibitory concentration (MIC) determination associated with therapeutic drug monitoring (TDM) and their combined interpretation are required. The objective of this narrative review is thus to suggest microbiological, pharmacological, and/or clinical situations for which this approach could be useful. Regarding the microbiological aspect, such as the detection of antibiotic resistance and its level, the preservation of broad-spectrum ß-lactams is particularly discussed. PK-PD profiles are relevant for difficult-to-reach infections and specific populations such as intensive care patients, cystic fibrosis patients, obese, or elderly patients. Finally, MIC and TDM are tools available to clinicians, who should not hesitate to use them to manage their patients.

Automated HPLC-MS/MS assay for the simultaneous determination of ten plasma antibiotic concentrations.

Therapeutic drug monitoring (TDM) of antibiotics (ATB) in patients with serious bacterial infections allows optimization of the efficacy of the treatment while reducing the risk of toxicity. Notably, early measurement of plasma beta-lactam concentration has been shown to be associated with reduced mortality in intensive care patients. In this context, a rapid, robust, and accurate assay method is essential for daily TDM. A fully automated procedure for quantification of the plasma concentrations of ten ATB was developed. The ATB were divided into two calibration pools, with Pool 1: aztreonam, ceftobiprole, cefoxitin, avibactam, tazobactam and Pool 2: metronidazole, ceftriaxone, daptomycin, ceftolozane, moxifloxacin. Sample preparation consisting of acetonitrile plasma protein precipitation and H20 dilution was applied to all analytes. This procedure was carried out by an automated sample preparation system directly coupled to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Since the instrument extracts sample n while sample n-1 is in the LC-MS/MS system, the delay between obtaining the results for two samples corresponds to the analytical run time, which is less than 7 min. The method was validated according to the Food and Drug Administration guidelines. The method was sensitive (lower limit of quantification 0.1-1 mg/L, depending on the ATB), accurate (intra/inter-assay bias -14.8 to 14.2 %) and precise (intra/inter-assay CVs 1.27 to 16.3 %). Application of the TDM assay was illustrated by the report of an intensive care patient treated with the ceftazidime/aztreonam/avibactam combination. Four assays were performed in 8 days with results returned within 24 h to quickly manage the dose regimen in this patient. An automated, simple, rapid, robust LC-MS/MS analysis was developed and validated for the simultaneous quantification of plasma concentrations of 10 ATB and was applied with success to perform TDM. This method provides a shorter turnaround time than classic sample batch-based analytical methods.

Vaginal microbiota as an unusual cause of spondylodiscitis.

The incidence of extra pelvic infections due to vaginal microflora bacteria has increased as growth media and methods of isolation have improved. However, bone infections seem to be still relatively rare, and little is known about their risk factors, clinical presentation, treatment and final outcome. We describe here a spondylodiscitis due to Gardnerella vaginalis, Atopobium vaginae, Peptostreptococcus indolicus and Prevotella amnii, anaerobic bacteria from vaginal microbiota. Our patient had no obvious predisposing factor and recovered after antibiotic treatment. To our knowledge, this case is the first reported spondylodiscitis caused by polymicrobial vaginal flora in a healthy, immunocompetent woman.

Antimicrobial Resistance in Enterobacterales Recovered from Urinary Tract Infections in France.

In the context of increasing antimicrobial resistance in Enterobacterales, the management of these UTIs has become challenging. We retrospectively assess the prevalence of antimicrobial resistance in Enterobacterales isolates recovered from urinary tract samples in France, between 1 September 2017, to 31 August 2018. Twenty-six French clinical laboratories provided the susceptibility of 134,162 Enterobacterales isolates to 17 antimicrobials. The most frequent species were E. coli (72.0%), Klebsiella pneumoniae (9.7%), Proteus mirabilis (5.8%), and Enterobacter cloacae complex (2.9%). The overall rate of ESBL-producing Enterobacterales was 6.7%, and ranged from 1.0% in P. mirabilis to 19.5% in K. pneumoniae, and from 3.1% in outpatients to 13.6% in long-term care facilities. Overall, 4.1%, 9.3% and 10.5% of the isolates were resistant to cefoxitin, temocillin and pivmecillinam. Cotrimoxazole was the less active compound with 23.4% resistance. Conversely, 4.4%, 12.9%, and 14.3% of the strains were resistant to fosfomycin, nitrofurantoin, and ciprofloxacin. However, less than 1% of E. coli was resistant to fosfomycin and nitrofurantoin. We identified several trends in antibiotics resistances among Enterobacterales isolates recovered from the urinary tract samples in France. Carbapenem-sparing drugs, such as temocillin, mecillinam, fosfomycin, cefoxitin, and nitrofurantoin, remained highly active, including towards ESBL-E.

Diagnosis in France of a Non-Toxigenic tox Gene-Bearing Strain of Corynebacterium diphtheriae in a Young Male Back From Senegal.

Cutaneous diphtheria is uncommon in Europe. In this study, we report a case of imported cutaneous infection due to a non-toxigenic but tox gene-bearing (NTTB) strain of Corynebacterium diphtheriae. The NTTB strains are recognized as emerging pathogens across Europe, and physicians and bacteriologists should be aware of the circulation of these strains.

Prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae infections detected by real-time PCR among individuals reporting sexual assaults in the Paris, France area.

BACKGROUND: Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) are the most common bacteria involved in sexually transmitted infections (STIs). In France, combined screening for CT and NG using nucleic acid amplification tests is recommended in populations that are considered at risk. However, no data have been published about victims of sexual assaults. The aim of this retrospective study was to assess the usefulness of real-time PCR for the rapid detection of CT and NG genomic DNA, and the prevalence of CT/NG infections in a sample of sexual assault victims examined at a department of forensic medicine. METHODS: Between July 2012 and July 2013, 326 adults and adolescents aged over 12 years reported a sexual assault and they were referred to the Department of Forensic Medicine for a medical and forensic examination. Secretions from urogenital (cervix or vagina), anorectal, or pharyngeal sites were collected for CT/NG assays. RESULTS: CT and NG were detected in 48/326 (15%) and 16/326 patients (5%), respectively, where 10 (3%) had a CT/NG co-infection. Among 48 patients with CT infection, 13 (27.1%) patients had co-occurring genital and anorectal infections. For the pharyngeal sites, 3/21 men (14.3%) were NG-positive. CONCLUSIONS: Given the high prevalence of CT and NG infections, systematic screening of both pathogens at the time of forensic examination should provide an opportunity for the early treatment of diagnosed STIs.

Phylogenetic, virulence and antibiotic resistance characteristics of commensal strain populations of Escherichia coli from community subjects in the Paris area in 2010 and evolution over 30 years.

It is important to study commensal populations of Escherichia coli because they appear to be the reservoir of both extra-intestinal pathogenic E. coli and antibiotic resistant strains of E. coli. We studied 279 dominant faecal strains of E. coli from 243 adults living in the community in the Paris area in 2010. The phylogenetic group and subgroup [sequence type complex (STc)] of the isolates and the presence of 20 virulence genes were determined by PCR assays. The O-types and resistance to 18 antibiotics were assessed phenotypically. The B2 group was the most frequently recovered (34.0 %), followed by the A group (28.7 %), and other groups were more rare. The most prevalent B2 subgroups were II (STc73), IV (STc141), IX (STc95) and I (STc131), with 22.1, 21.1, 16.8 and 13.7 %, respectively, of the B2 group strains. Virulence factors (VFs) were more common in B2 group than other strains. One or more resistances were found in 125 strains (44.8 % of the collection) but only six (2.2 % of the collection) were multiresistant; no extended-spectrum beta-lactamase-producing strain was isolated. The C phylogroup and clonal group A strains were the most resistant. No trade-off between virulence and resistance was evidenced. We compared these strains with collections of strains gathered under the same conditions 30 and 10 years ago. There has been a parallel and linked increase in the frequency of B2 group strains (from 9.4 % in 1980, to 22.7 % in 2000 and 34.0 % in 2010) and of VFs. Antibiotic resistance also increased, from 22.6 % of strains resistant to at least one antibiotic in 1980, to 31.8 % in 2000 and 44.8 % in 2010; resistance to streptomycin, however, remained stable. Commensal human E. coli populations have clearly evolved substantially over time, presumably reflecting changes in human practices, and particularly increasing antibiotic use.

Benefits of Rapid Molecular Diagnosis of Chlamydia Trachomatis and Neisseria Gonorrhoeae Infections in Women Attending Family Planning Clinics.

We evaluated the benefits of on-demand systematic screening for Chlamydia trachomatis and Neisseria gonorrhoeae using the Xpert CT/NG assay in 589 women attending family planning clinics. The sexually transmitted infection prevalence was 16.5% with 15.1% C. trachomatis and 3.1% N. gonorrhoeae infections. The on-demand test allowed for a quicker management of patients at high risk for sexually transmitted infections.

Real-time PCR for quantitative analysis of human commensal Escherichia coli populations reveals a high frequency of subdominant phylogroups.

Escherichia coli is divided into four main phylogenetic groups, which each exhibit ecological specialization. To understand the population structure of E. coli in its primary habitat, we directly assessed the relative proportions of these phylogroups from the stools of 100 healthy human subjects using a new real-time PCR method, which allows a large number of samples to be studied. The detection threshold for our technique was 0.1% of the E. coli population, i.e., 10(5) CFU/g of feces; in other methods based on individual colony analysis, the threshold is 10%. One, two, three, or four phylogenetic groups were simultaneously found in 21%, 48%, 21%, and 8% of the subjects, respectively. Phylogroups present at a threshold of less than 10% of the population were found in 40% of the subjects, revealing high within-individual diversity. Phylogroups A and B2 were detected in 74% and 70% of the subjects, respectively; phylogroups B1 and D were detected in 36% and 32%, respectively. When phylogroup B2 was dominant, it tended not to cooccur with other phylogroups. In contrast, other phylogroups were present when phylogroup A was dominant. These data indicate a complex pattern of interactions between the members of a single species within the human gut and identify a reservoir of clones that are present at a low frequency. The presence of these minor clones could explain the fluctuation in the composition of the E. coli microbiota within single individuals that may be seen over time. They could also constitute reservoirs of virulent and/or resistant strains.

Postsurgical meningitis due to multiresistant Acinetobacter baumannii successfully treated with high doses of ampicillin/sulbactam combined with rifampicin and fosfomycin.

We report a case of postsurgical meningitis caused by multiresistant Acinetobacter baumannii successfully treated with high doses of ampicillin/sulbactam combined with rifampicin and fosfomycin.