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.

Optimized blood culture strategy to document febrile neutropenia.

BACKGROUND: Poor and delayed microbiological documentation of episodes of febrile neutropenia (EFN) deserves improvement. We assessed the impact of a new blood culture (BC) sampling protocol to optimize the diagnosis of bloodstream infection during EFN, compared with standard of care protocol. METHODS: This pre/post intervention included patients who presented an EFN in a pediatric hematology-oncology center. Data were compared between 1-year periods P1 (110 EFN, 53 patients) and P2 (124 EFN, 53 patients). Pre-intervention settings were 1-2 mL of blood cultured per BC set and several samplings over days (multisampling strategy) during period P1 vs. one unique early sampling of a large volume of blood (0.5-60 mL) depending on patient weight during period P2 (single-sampling weight-adapted strategy). Microbial detection and time-to-diagnosis were evaluated. RESULTS: Seventeen EFNs were microbiologically documented in P1 (15.5%) and 26 in P2 (21%). The rate of positive BC sets increased during P2 (10.4% vs. 5.8%). All cases of bacteremia were documented by BC drawn during the first 4 days of fever, and during P2 by samples obtained on the first day of fever. CONCLUSIONS: Bacteremia detection was improved. This proof-of-concept study shows benefits of combining the single-sampling strategy with large weight-adapted blood sampling strategy during EFN. IMPACT: Combination of single-sampling and weight-adapted blood culture strategies showed benefits in the documentation of bloodstream infections during febrile neutropenia. Bacteremia detection was improved in this preliminary study and this warrants further evaluation in the overall pediatric population. We observed no adverse effects associated with the new strategy while overall blood sparing was improved and handling of intravascular devices was reduced. The good tolerance of the blood sampling suggests that the recommended 1% volume limitation in children could be reconsidered. A similar evaluation is justified in the overall pediatric population suspected for bloodstream infection.

Comparative Whole-Genome Phylogeny of Animal, Environmental, and Human Strains Confirms the Genogroup Organization and Diversity of the Stenotrophomonas maltophilia Complex.

The Stenotrophomonas maltophilia complex (Smc) comprises opportunistic environmental Gram-negative bacilli responsible for a variety of infections in both humans and animals. Beyond its large genetic diversity, its genetic organization in genogroups was recently confirmed through the whole-genome sequencing of human and environmental strains. As they are poorly represented in these analyses, we sequenced the whole genomes of 93 animal strains to determine their genetic background and characteristics. Combining these data with 81 newly sequenced human strains and the genomes available from RefSeq, we performed a genomic analysis that included 375 nonduplicated genomes with various origins (animal, 104; human, 226; environment, 30; unknown, 15). Phylogenetic analysis and clustering based on genome-wide average nucleotide identity confirmed and specified the genetic organization of Smc in at least 20 genogroups. Two new genogroups were identified, and two previously described groups were further divided into two subgroups each. Comparing the strains isolated from different host types and their genogroup affiliation, we observed a clear disequilibrium in certain groups. Surprisingly, some antimicrobial resistance genes, integrons, and/or clusters of attC sites lacking integron-integrase (CALIN) sequences targeting antimicrobial compounds extensively used in animals were mainly identified in animal strains. We also identified genes commonly found in animal strains coding for efflux systems. The result of a large whole-genome analysis performed by us supports the hypothesis of the putative contribution of animals as a reservoir of Stenotrophomonas maltophilia complex strains and/or resistance genes for strains in humans.IMPORTANCE Given its naturally large antimicrobial resistance profile, the Stenotrophomonas maltophilia complex (Smc) is a set of emerging pathogens of immunosuppressed and cystic fibrosis patients. As it is group of environmental microorganisms, this adaptation to humans is an opportunity to understand the genetic and metabolic selective mechanisms involved in this process. The previously reported genomic organization was incomplete, as data from animal strains were underrepresented. We added the missing piece of the puzzle with whole-genome sequencing of 93 strains of animal origin. Beyond describing the phylogenetic organization, we confirmed the genetic diversity of the Smc, which could not be estimated through routine phenotype- or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF)-based laboratory tests. Animals strains seem to play a key role in the diversity of Smc and could act as a reservoir for mobile resistance genes. Some genogroups seem to be associated with particular hosts; the genetic support of this association and the role of the determinants/corresponding genes need to be explored.

An unusual Staphylococcus saccharolyticus spondylodiscitis post kyphoplasty: a case report.

BACKGROUND: Staphylococcus saccharolyticus is a rarely encountered coagulase-negative, which grows slowly and its strictly anaerobic staphylococcus from the skin. It is usually considered a contaminant, but some rare reports have described deep-seated infections. Virulence factors remain poorly known, although, genomic analysis highlights pathogenic potential. CASE PRESENTATION: We report a case of Staphylococcus saccharolyticus spondylodiscitis that followed kyphoplasty, a procedure associated with a low rate but possible severe infectious complication (0.46%), and have reviewed the literature. This case specifically stresses the risk of healthcare-associated S. saccharolyticus infection in high-risk patients (those with a history of alcoholism and heavy smoking). CONCLUSION: S. saccharolyticus infection is difficult to diagnose due to microbiological characteristics of this bacterium; it requires timely treatment, and improved infection control procedure should be encouraged for high-risk patients.

Are animals a source of Stenotrophomonas maltophilia in human infections? Contributions of a nationwide molecular study.

Stenotrophomonas maltophilia (Sm) is an archetypal environmental opportunistic bacterium responsible for health care-associated infections. The role of animals in human Sm infections is unknown. This study aims to reveal the genetic and phylogenetic relationships between pathogenic strains of Sm, both animal and human, and identify a putative role for animals as a reservoir in human infection. We phenotypically and genotypically characterized 61 Sm strains responsible for animal infections (mainly respiratory tract infections in horses) from a French nationwide veterinary laboratory network. We tested antimicrobial susceptibility and performed MLST and genogrouping using the concatenation of the seven housekeeping genes from the original MLST scheme. Excluding the eight untypeable strains owing to the lack of gene amplification, only 10 out of the 53 strains yielded a known ST (ST5, ST39, ST162, ST8, ST27, ST126, ST131). The genogroup distribution highlighted not only genogroups (genogroups 5 and 9) comprised exclusively of animal strains but also genogroups shared by human and animal strains. Interestingly, these shared genogroups were primarily groups 2 and 6, which have previously been identified as the two most frequent genogroups among human-pathogenic Sm strains, especially among respiratory pathogens. The antimicrobial susceptibility testing underlined the presence of acquired resistance: 18.8 and 7.5% of the tested isolates were resistant to the sulfonamide-trimethoprim combination and ciprofloxacin, respectively. Animal strains of Sm shared phylogenetic traits with some of the most successful human strains. The exact relationships between the human and animal strains, and the genetic support of these common traits, need to be determined.

Osteomyelitis of the jaw: time to rethink the bone sampling strategy?

This work aims at describing the diversity of osteomyelitis of the jaw (OJ) and at assessing the relevance of a new method designed to avoid salivary contamination during bone sampling in order to improve microbiological analysis and clinical decision-making. We reviewed medical and microbiological data of patients with a suspected OJ based on clinical and/or CT-scan signs and at least one bone sample made for microbiological analysis. During the study period, a new procedure for intraoral bone sampling was elaborated by surgeons and infectious diseases specialists authoring this article (based on stratified samples, cleaning of the surgical site and change of instruments between each sample). A comparison of the microbiological analyses between the two procedures was performed. From 2012 to 2017, 56 patients were included. Median age was 58 years (11-90), sex ratio: 1.24. Main risk factors were having a dental disease (n = 24) or cancer (n = 21). Nineteen patients with the new sample procedure were compared to 37 patients with standard procedure, especially non-cancer patients (n = 16 and 19, respectively). With the new procedure, a median of 3 (1-7) microorganisms per sample was recovered, vs. 7 (1-14) with the former (p < 0.001), a significant decrease of the microbial density was observed for all types of microbes, especially in deeper samples and cultures were more frequently sterile. The way sampling is managed deeply influences microbiological analysis. This strategy facilitates the distinction between pathogens and contaminants and should constitute the first step toward an evidence-based antimicrobial strategy for OJ.

Mycobacterium bourgelatii sp. nov., a rapidly growing, non-chromogenic species isolated from the lymph nodes of cattle.

Three independent strains of a rapidly growing, non-chromogenic member of the genus Mycobacterium were isolated from lymph nodes of French cattle. Identification of the isolates was carried out using a polyphasic approach. The nearly complete SSU rRNA gene sequences (>1200 bp) of the strains MLB-A23, MLB-A30 and MLB-A84(T) were identical. A phylogenetic analysis of these unique SSU rRNA gene sequences showed that these strains were most closely related to Mycobacterium intermedium. Further phylogenetic analysis based on concatenated sequences (2854 bp) of four housekeeping genes (hsp65, rpoB, sodA and tuf), the transfer-messenger RNA (tmRNA) and SSU rRNA genes indicated that these three strains represented a distinct species that shares a common ancestor with M. intermedium. Phylogenetic and phenotypic data strongly indicate that the strains MLB-A23, MLB-A30 and MLB-A84(T) belong to a novel mycobacterial species for which the name Mycobacterium bourgelatii sp. nov. is proposed. The type strain is MLB-A84(T) ( = CIP 110557(T) = DSM 45746(T)).

Drosophila melanogaster Systemic Infection Model to Study Altered Virulence during Polymicrobial Infection by Aeromonas.

BACKGROUND: Polymicrobial infections are complex infections associated with worse outcomes compared to monomicrobial infections. We need simple, fast, and cost-effective animal models to assess their still poorly known pathogenesis. METHODS: We developed a Drosophila melanogaster polymicrobial infection model for opportunistic pathogens and assessed its capacity to discriminate the effects of bacterial mixtures taken from cases of human polymicrobial infections by Aeromonas strains. A systemic infection was obtained by needle pricking the dorsal thorax of the flies, and the fly survival was monitored over time. Different lineages of the flies were infected by a single strain or paired strains (strain ratio 1:1). RESULTS: Individual strains killed more than 80% of the flies in 20 h. The course of infection could be altered with a microbial mix. The model could distinguish between the diverse effects (synergistic, antagonistic, and no difference) that resulted in a milder, more severe, or similar infection, depending on the paired strain considered. We then investigated the determinants of the effects. The effects were maintained in deficient fly lineages for the main signaling pathways (Toll deficient and IMD deficient), which suggests an active microbe/microbe/host interaction. CONCLUSION: These results indicate that the D. melanogaster systemic infection model is consistent with the study of polymicrobial infection.

Lesson from the COVID-19 pandemic lockdown: A major change of hospital-diagnosed bacteremia epidemiology.

OBJECTIVES: When the COVID-19 pandemic reached France early in 2020, the enforced nationwide lockdown deeply altered lifestyle as well as hospital processes and modalities of care. The aim of the study was to evaluate the impact during the lockdown of the first epidemic wave on the epidemiology of bacteremia in one French University Hospital. PATIENTS AND METHODS: Retrospective cohort study including adult patients with positive blood culture between 23rd March to 24th May 2020. The clinical-microbiological characteristics were compared with those of the period from 25th March to 26th May 2019. The data were adjusted to the number of hospitalizations (h). RESULTS: In 2020, 189 bacteremia were diagnosed from 1939 vials (9658 hospitalizations, 10911 emergency room consultations) compared to 143 from 1976 vials (14797 hospitalizations, 16493 emergency room consultations) recorded in 2019. The incidence of bacteremia increased up to 19.7 per 1000h in 2020 vs 9.7 in 2019 (p < 0.001). The main differences (2020 vs 2019) were: Staphylococcus aureus bacteremia (2.4 vs 1.0/1000h, p = 0.012), polymicrobial bacteremia (2.2 vs 0.9/1000h p = 0.013) and Gram-negative bacteremia (8.9 vs 4.3/1000h, p < 0.01). Conversely, Streptococcus pneumoniae incidence decreased (0 vs 0.47/1000h, p = 0.047). The standardized incidence ratio calculation confirmed these results. CONCLUSION: The lockdown and the impact of the first wave of the Covid-19 pandemic on the health system resulted in increased hospital-diagnosed bacteremia and decreased pneumococcal bacteremia. Disruption and overload of ICUs, lockdown with preventive control measures, and decrease in human-to-human interaction may have been the main reasons.

A Global Survey of Hypervirulent Aeromonas hydrophila (vAh) Identified vAh Strains in the Lower Mekong River Basin and Diverse Opportunistic Pathogens from Farmed Fish and Other Environmental Sources.

Hypervirulent Aeromonas hydrophila (vAh) has emerged as the etiologic agent of epidemic outbreaks of motile Aeromonas septicemia (MAS) in high-density aquaculture of farmed carp in China and catfish in the United States, which has caused millions of tons of lost fish. We conducted a global survey to better understand the evolution, geographical distribution, and phylogeny of vAh. Aeromonas isolates were isolated from fish that showed clinical symptoms of MAS, and pure cultures were screened for the ability to utilize myo-inositol as the sole carbon source. A total of 113 myo-inositol-utilizing bacterial strains were included in this study, including additional strains obtained from previously published culture collections. Based on a gyrB phylogeny, this collection included 66 A. hydrophila isolates, 48 of which were vAh. This collection also included five new vAh isolates from diseased Pangas catfish (Pangasius pangasius) and striped catfish (Pangasianodon hypophthalmus) obtained in Cambodia and Vietnam, respectively. Genome sequences were generated from representative vAh and non-vAh isolates to evaluate the potential for lateral genetic transfer of the myo-inositol catabolism pathway. Phylogenetic analyses of each of the nine genes required for myo-inositol utilization revealed the close affiliation of vAh strains regardless of geographic origin and suggested lateral genetic transfer of this catabolic pathway from an Enterobacter species. Prediction of virulence factors was conducted to determine differences between vAh and non-vAh strains in terms of virulence and secretion systems. Core genome phylogenetic analyses on vAh isolates and Aeromonas spp. disease isolates (55 in total) were conducted to evaluate the evolutionary relationships among vAh and other Aeromonas sp. isolates, which supported the clonal nature of vAh isolates. IMPORTANCE This global survey of vAh brought together scientists that study fish disease to evaluate the evolution, geographical distribution, phylogeny, and hosts of vAh and other Aeromonas sp. isolates. In addition to vAh isolates from China and the United States, four new vAh isolates were isolated from the lower Mekong River basin in Cambodia and Vietnam, indicating the significant threat of vAh to modern aquaculture and the need for improved biosecurity to prevent vAh spread.

Multilocus genetics to reconstruct aeromonad evolution.

BACKGROUND: Aeromonas spp. are versatile bacteria that exhibit a wide variety of lifestyles. In an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics in terms of genetic diversity, population structure and mode of evolution among Aeromonas spp. A collection of 195 Aeromonas isolates from human, animal and environmental sources was therefore genotyped using multilocus sequence analysis (MLSA) based on the dnaK, gltA, gyrB, radA, rpoB, tsf and zipA genes. RESULTS: The MLSA showed a high level of genetic diversity among the population, and multilocus-based phylogenetic analysis (MLPA) revealed 3 major clades: the A. veronii, A. hydrophila and A. caviae clades, among the eleven clades detected. Lower genetic diversity was observed within the A. caviae clade as well as among clinical isolates compared to environmental isolates. Clonal complexes, each of which included a limited number of strains, mainly corresponded to host-associated subsclusters of strains, i.e., a fish-associated subset within A. salmonicida and 11 human-associated subsets, 9 of which included only disease-associated strains. The population structure was shown to be clonal, with modes of evolution that involved mutations in general and recombination events locally. Recombination was detected in 5 genes in the MLSA scheme and concerned approximately 50% of the STs. Therefore, these recombination events could explain the observed phylogenetic incongruities and low robustness. However, the MLPA globally confirmed the current systematics of the genus Aeromonas. CONCLUSIONS: Evolution in the genus Aeromonas has resulted in exceptionally high genetic diversity. Emerging from this diversity, subsets of strains appeared to be host adapted and/or "disease specialized" while the A. caviae clade displayed an atypical tempo of evolution among aeromonads. Considering that A. salmonicida has been described as a genetically uniform pathogen that has adapted to fish through evolution from a variable ancestral population, we hypothesize that the population structure of aeromonads described herein suggested an ongoing process of adaptation to specialized niches associated with different degrees of advancement according to clades and clusters.

Aeromonas: the multifaceted middleman in the One Health world.

Aeromonas is at the interface of all the One Health components and represents an amazingly sound test case in the One Health approach, from economic loss in aquaculture tochallenges related to antibiotic-resistant bacteria selected from the environment. In human health, infections following leech therapy is an outstanding example of such One Health challenges. Aeromonads are not only ubiquitous environmental bacteria, able to rapidly colonize and cause opportunistic infections in humans and animals, they are also capable of promoting interactions and gene exchanges between the One Health components. This makes this genus a key amplifier of genetic transfer, especially of antibiotic resistance genes.

Performance of a New MicroScan WalkAway PC30 panel and disk diffusion method for detection of oxacillin resistance in Staphylococcus spp.

The performance of the MicroScan WalkAway PC30 panel for detection of oxacillin resistance was evaluated by use of a collection of 420 staphylococcus isolates. The addition of a cefoxitin test (4 mg/liter) to the oxacillin MIC determination increased its raw performance for Staphylococcus aureus; additional data were required for coagulase-negative staphylococci.

Validation of a partial rpoB gene sequence as a tool for phylogenetic identification of aeromonads isolated from environmental sources.

A collection of 50 aeromonads isolated from environmental sources were studied, together with all known Aeromonas nomenspecies, by phenotypic, amplified 16S rDNA restriction analysis (16S rDNA RFLP) and by partial sequence alignment of both 16S rDNA and rpoB genes. Although most of the type strain showed a unique phenotypic pattern, a database constructed on type strain phenotype allowed the identification of only 24% of the isolates. Analysis of 16S rDNA RFLP and the rpoB sequence were almost concordant in identifying environmental isolates at the species level, except for strains belonging to Aeromonas caviae spp., which were not differentiated from Aeromonas aquariorum, nor Aeromonas hydrophila susbsp. dhakensis by 16S rDNA RFLP. In addition, rpoB gene analysis clustered separately a group of isolates found in snails within the A. hydrophila species. In contrast to 16S rDNA RFLP and rpoB, the partial 16S rDNA sequence analysis was weak in resolving species identity. Part of these results, phenotypic and phylogenetic data, showed that Aeromonas molluscorum and Aeromonas sharmana are distant from all other Aeromonas species and that the type species of A. hydrophila subsp. anaerogenes is similar to A. caviae and should be considered synonymous.

Bloodstream infections - Standard and progress in pathogen diagnostics.

BACKGROUND: Bloodstream infection (BSI) is a major public health burden worldwide, with high mortality. Patient outcome is critically influenced by delayed therapy, and fast and accurate pathogen diagnostics decisively improves the care of patients. During the past two decades major improvements have been made in the diagnostic performance of blood culture diagnostics through actions on pre-analysis and time to result. AIMS: To review and discuss the literature for standard procedures and the progress in BSI pathogen diagnostics, and to propose a new mindset to reach an improved diagnostic workflow. SOURCES: Scientific articles and reviews available through NCBI/Pubmed. CONTENT: Blood culture performance relies largely on the quality of its pre-analytical phase that is improved with educational actions monitored by using key performance indicators, and external quality assessment. Advanced blood culture systems now provide tools for an automated estimation of bottle filling. These proved efficient to facilitate effective training for improving blood collection. On analytic aspects, rapid methods for pathogen identification, among which matrix-assisted laser desorption/ionization time of flight mass spectrometry dominates, and rapid antimicrobial susceptibility testing are reviewed. These technical developments call for improvements in all other steps, especially in pre- and post-analytic logistics to give the full reciprocation of these techniques on patient management. This aspect is summarized by the term 'microbiologistics', which covers all possible improvements in the logistic chain from sampling to report. IMPLICATIONS: Progress in BSI pathogen diagnostics is based on a bundle approach that includes optimization of the pre-analytical parameters, rapid start of incubation, the use of rapid methods, re-organization (e.g. 24/7, transportation service) and a close involvement of antimicrobial stewardship teams. These developments lead to define a new standard for bloodstream infection diagnostics.

Comparative and Evolutionary Genomics of Isolates Provide Insight into the Pathoadaptation of Aeromonas.

Aeromonads are ubiquitous aquatic bacteria that cause opportunistic infections in humans, but their pathogenesis remains poorly understood. A pathogenomic approach was undertaken to provide insights into the emergence and evolution of pathogenic traits in aeromonads. The genomes of 64 Aeromonas strains representative of the whole genus were analyzed to study the distribution, phylogeny, and synteny of the flanking sequences of 13 virulence-associated genes. The reconstructed evolutionary histories varied markedly depending on the gene analyzed and ranged from vertical evolution, which followed the core genome evolution (alt and colAh), to complex evolution, involving gene loss by insertion sequence-driven gene disruption, horizontal gene transfer, and paraphyly with some virulence genes associated with a phylogroup (aer, ser, and type 3 secretion system components) or no phylogroup (type 3 secretion system effectors, Ast, ExoA, and RtxA toxins). The general pathogenomic overview of aeromonads showed great complexity with diverse evolution modes and gene organization and uneven distribution of virulence genes in the genus; the results provided insights into aeromonad pathoadaptation or the ability of members of this group to emerge as pathogens. Finally, these findings suggest that aeromonad virulence-associated genes should be examined at the population level and that studies performed on type or model strains at the species level cannot be generalized to the whole species.

Prospective nationwide study of Aeromonas infections in France.

We report a systematic prospective multicenter nationwide study of clinical Aeromonas infections in France. During 6 months (May to October 2006), 78 cases of aeromonosis were reviewed for risk factors and clinical, microbiological, and antimicrobial susceptibility data. They included wound infections (44%), bacteremia (26%), enteritis (19%), respiratory tract infections (6%), and miscellaneous (5%) infections.

Leech management before application on patient: a nationwide survey of practices in French university hospitals.

Background: Leech therapy in plastic/reconstructive microsurgery significantly improves a successful outcome of flap salvage but the drawback is a risk of severe infection that results in a drop of the salvage rates from 70-80% to below 30%. We report the results of a national survey conducted in all the French university hospitals to assess the current extent of use of leech for medical practices in the hospital and to investigate maintenance, delivery practices and prevention of the risk of infection. Methods: Data concerning conditions of storage, leech external decontamination, microbiological controls, mode of delivery and antibiotic prophylaxis were collected from all the French university hospitals in practicing leech therapy, on the basis of a standardized questionnaire. Results: Twenty-eight of the 32 centers contacted filled the questionnaire, among which 23 practiced leech therapy, mostly with a centralized storage in the pharmacy; 39.1% of the centers declared to perform leech external decontamination and only 2 centers recurrent microbiological controls of the water storage. Leech delivery was mostly nominally performed (56.5%), but traceability of the leech batch number was achieved in only 39.1% of the cases. Only 5 centers declared that a protocol of antibiotic prophylaxis was systematically administered during leech therapy: either quinolone (2), sulfamethoxazole/trimethoprim (2) or amoxicillin/clavulanic acid (1). Conclusions: Measures to prevent infectious complications before application to patient have to be better applied and guidelines of good practices are necessary.

Genome-driven evaluation and redesign of PCR tools for improving the detection of virulence-associated genes in aeromonads.

Many virulence factors have been described for opportunistic pathogens within the genus Aeromonas. Polymerase Chain Reactions (PCRs) are commonly used in population studies of aeromonads to detect virulence-associated genes in order to better understand the epidemiology and emergence of Aeromonas from the environment to host, but their performances have never been thoroughly evaluated. We aimed to determine diagnostic sensitivity and specificity of PCR assays for the detection of virulence-associated genes in a collection of Aeromonas isolates representative for the genetic diversity in the genus. Thirty-nine Aeromonas strains belonging to 27 recognized species were screened by published PCR assays for virulence-associated genes (act, aerA, aexT, alt, ascFG, ascV, ast, lafA, lip, ser, stx1, stx2A). In parallel, homologues of the 12 putative virulence genes were searched from the genomes of the 39 strains. Of the 12 published PCR assays for virulence factors, the comparison of PCR results and genome analysis estimated diagnostic sensitivities ranging from 34% to 100% and diagnostic specificities ranged from 71% to 100% depending upon the gene. To improve the detection of virulence-associated genes in aeromonads, we have designed new primer pairs for aerA/act, ser, lafA, ascFG and ascV, which showed excellent diagnostic sensitivity and specificity. Altogether, the analysis of high quality genomic data, which are more and more easy to obtain, provides significant improvements in the genetic detection of virulence factors in bacterial strains.

Low-Level Antimicrobials in the Medicinal Leech Select for Resistant Pathogens That Spread to Patients.

Fluoroquinolones (FQs) and ciprofloxacin (Cp) are important antimicrobials that pollute the environment in trace amounts. Although Cp has been recommended as prophylaxis for patients undergoing leech therapy to prevent infections by the leech gut symbiont Aeromonas, a puzzling rise in Cp-resistant (Cpr) Aeromonas infections has been reported. We report on the effects of subtherapeutic FQ concentrations on bacteria in an environmental reservoir, the medicinal leech, and describe the presence of multiple antibiotic resistance mutations and a gain-of-function resistance gene. We link the rise of CprAeromonas isolates to exposure of the leech microbiota to very low levels of Cp (0.01 to 0.04 µg/ml), <1/100 of the clinical resistance breakpoint for Aeromonas Using competition experiments and comparative genomics of 37 strains, we determined the mechanisms of resistance in clinical and leech-derived Aeromonas isolates, traced their origin, and determined that the presence of merely 0.01 µg/ml Cp provides a strong competitive advantage for Cpr strains. Deep-sequencing the Cpr-conferring region of gyrA enabled tracing of the mutation-harboring Aeromonas population in archived gut samples, and an increase in the frequency of the Cpr-conferring mutation in 2011 coincides with the initial reports of CprAeromonas infections in patients receiving leech therapy.IMPORTANCE The role of subtherapeutic antimicrobial contamination in selecting for resistant strains has received increasing attention and is an important clinical matter. This study describes the relationship of resistant bacteria from the medicinal leech, Hirudo verbana, with patient infections following leech therapy. While our results highlight the need for alternative antibiotic therapies, the rise of Cpr bacteria demonstrates the importance of restricting the exposure of animals to antibiotics approved for veterinary use. The shift to a more resistant community and the dispersion of Cpr-conferring mechanisms via mobile elements occurred in a natural setting due to the presence of very low levels of fluoroquinolones, revealing the challenges of controlling the spread of antibiotic-resistant bacteria and highlighting the importance of a holistic approach in the management of antibiotic use.