A long-term survey of Serratia spp. bloodstream infections revealed an increase of antimicrobial resistance involving adult population.

Serratia spp. is a well-recognized pathogen in neonates; however, limited data are available in adults. We studied microbiological and clinical characteristics of Serratia spp. causing bloodstream infections (BSI) in our institution (January 2005-July 2020). Overall, 141 BSI episodes affecting 139 patients were identified and medical records reviewed. Antimicrobial susceptibility was recovered from our informatics system and 118 isolates from 116 patients were available for further microbiological studies. Whole genome sequencing (WGS) was completed in 107 isolates. Incidence of Serratia BSI was 0.3/1000 overall admissions (range 0.12-0.60), with maximum prevalence (27 episodes, 19.1%) during 2017-2018. Relevant patients' clinical characteristics were 71.9% ≥60 years (n = 100), with high comorbidity rates (49%, ≥2), 23 (74.2%) of them died within 1 month of the BSI episode. WGS identified all isolates as Serratia marcescens when Kraken bioinformatics taxonomic tool was used despite some which were identified as Serratia nematodiphila (32/118) or Serratia ureilytica (5/118) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nevertheless, when using MASH distance, Serratia nevei (63/107), S. ureilytica (38/107), and S. marcescens (6/107) were assigned. Carbapenemase (blaVIM-1) and extended-spectrum ß-lactases (ESBL) (blaSHV-12) genes were found in seven and three isolates, respectively, one of them expressing both genes. The worldwide-disseminated IncL/M scaffold plasmid was identified in six VIM producers. Four genotypes were established based on their virulence factors and resistome. Serratia spp. emerged as a relevant nosocomial pathogen causing BSI in elderly patients in our hospital, particularly in recent years with a remarkable increase in antibiotic resistance. ESBL and carbapenemases production related to plasmid dissemination are particularly noteworthy.IMPORTANCESerratia spp. is the third most frequent pathogen involved in outbreaks at neonatal facilities and is primarily associated with bacteremia episodes. In this study, we characterized all causing bloodstream infection (BSI) in patients admitted to our hospital during a 16-year period (2005-2020). Despite having no neonatal intensive care unit in our hospital, this study revealed that Serratia spp. is a relevant pathogen causing BSI in elderly patients with high comorbidity rates. A significant increase of antimicrobial resistance was detected over time, particularly in 2020 and coinciding with the coronavirus disease (COVID-19) pandemic and nosocomial spread of multidrug-resistant Serratia spp. isolates. extended-spectrum ß-lactases and carbapenemases genes associated with plasmid dissemination, typically detected in other Enterobacterales species, were also identified, reinforcing the role of Serratia spp. in the antimicrobial resistance landscape. Additionally, this work highlights the need to reclassify the species of Serratia, since discrepancies were observed in the identification when using different tools.

Impact of Automated Blood Culture Systems on the Management of Bloodstream Infections: Results from a Crossover Diagnostic Clinical Trial.

Bloodstream infections are associated with high rates of morbidity and mortality. Blood culture remains the gold standard for the diagnosis of BSIs. We report a prospective crossover diagnostic clinical trial comparing the performances of two blood culture incubation systems: Virtuo and Bactec FX. The primary outcome was the time to detection (TTD) (from the loading of the sample into the incubator to the positivity signal). Patients over 16 years old suspected of having bacteremia/fungemia were included. They were divided into two strata with a total of 9,957 blood extractions. Initially, each stratum was randomly assigned to one of the incubators and then alternated every 2 weeks for 6 months. Each sample was inoculated into an aerobic bottle and an anaerobic bottle. All bottles were processed equally according to the laboratory's standard procedures after they were flagged positive. We analyzed 4,797 samples in the Virtuo system and 5,160 in the Bactec FX system. The median TTD was significantly lower for the Virtuo group (Virtuo, 15.2 h; Bactec FX, 16.3 h [P < 0.0001]). The turnaround time (TAT) (from sample loading to the Gram stain report) was also reduced with Virtuo (Virtuo, 26.2 h; Bactec FX, 28.3 h [P < 0.004]). When considering only samples from patients with antimicrobial treatment prior to blood culture extraction, the TTD was shorter for Virtuo (median differences in the TTD of 4.5 h for all bottles and 8.7 h for aerobic bottles only [P = 0.0001]). In conclusion, virtuo provided shorter TTD and TAT than Bactec FX. The difference in the median TTD was increased when considering samples incubated in aerobic bottles from patients with antimicrobial treatment. This could have an important effect on the faster diagnosis of BSIs. IMPORTANCE Bloodstream infections are associated with high rates of morbidity and mortality. Blood culture remains the gold standard for its diagnosis. While the identification of the pathogen and its antibiotic susceptibility is required to confirm the optimal antimicrobial regimen, reductions in the times to the detection of positivity and reporting of Gram stain results may be important and time-saving to reduce inappropriate antimicrobial use, improve patient outcomes, and decrease health care costs. We report the first clinical diagnostic study of this scale in a "real-world" setting with a crossover design, comparing two automatic blood culture incubators using samples from patients with a suspected diagnosis of bacteremia/sepsis, as opposed to spiked vials. Our study design mimics that of clinical trials performed for drug marketing authorization, but patient randomization was replaced with the crossover design. A shorter time to detection could have an important effect on the faster identification of causative microorganisms of BSIs and antimicrobial stewardship.

Real Life Clinical Impact of Antimicrobial Stewardship Actions on the Blood Culture Workflow from a Microbiology Laboratory.

BACKGROUND: Accelerating the diagnosis of bacteremia is one of the biggest challenges in clinical microbiology departments. The fast establishment of a correct treatment is determinant on bacteremic patients' outcomes. Our objective was to evaluate the impact of antimicrobial therapy and clinical outcomes of a rapid blood culture workflow protocol in positive blood cultures with Gram-negative bacilli (GNB). METHODS: A quasi-experimental before-after study was performed with two groups: (i) control group (conventional work-protocol) and (ii) intervention group (rapid workflow-protocol: rapid identification by Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) and antimicrobial susceptibility testing (AST) from bacterial pellet without overnight incubation). Patients were divided into different categories according to the type of intervention over treatment. Outcomes were compared between both groups. RESULTS: A total of 313 patients with GNB-bacteremia were included: 125 patients in the control group and 188 in the intervention. The time from positive blood culture to intervention on antibiotic treatment decreased from 2.0 days in the control group to 1.0 in the intervention group (p < 0.001). On the maintenance of correct empirical treatment, the control group reported 2.0 median days until the clinical decision, while in the intervention group was 1.0 (p < 0.001). In the case of treatment de-escalation, a significant difference between both groups (4.0 vs. 2.0, p < 0.001) was found. A decreasing trend on the change from inappropriate treatments to appropriate ones was observed: 3.5 vs. 1.5; p = 0.12. No significant differences were found between both groups on 7-days mortality or on readmissions in the first 30-days. CONCLUSIONS: Routine implementation of a rapid workflow protocol anticipates the report of antimicrobial susceptibility testing results in patients with GNB-bacteremia, decreasing the time to effective and optimal antibiotic therapy.

Epidemiology of the colonization and acquisition of methicillin-resistant staphylococci and vancomycin-resistant enterococci in dogs hospitalized in a clinic veterinary hospital in Spain.

Antibiotic resistance is one of the biggest threats to human and animal health. Methicillin-resistant Staphylococcus spp. (MRS) and vancomycin-resistant Enterococcus spp. (VRE) are of increasing importance in hospital and/or nosocomial infections and represent a potential risk of transmission to humans from infected or colonized companion animals. Studies on the risk factors associated with colonization by multiresistant bacteria in animals are scarce. The present study aimed to estimate the prevalence and incidence of MRS and VRE in canine patients hospitalized in a veterinary hospital and to identify the risk factors for its acquisition and persistence. Nasal and perianal swabs were obtained from 72 dogs. Antimicrobial susceptibility assays and molecular detection of mecA and van genes were performed. A prevalence of 13.9% and incidence of 26.5% was observed in dogs colonized by MRS at hospital admission and release, respectively, higher values than those described in most veterinary studies. Thirty-five Staphylococcus isolates had mecA gene and showed higher resistance levels to most of the antimicrobials evaluated. Previous and concomitant use of antibiotics and corticosteroids has been associated with an increase in MRS colonization. The use of antibiotics in other animals living with the canine patients has also been identified as an associated factor, suggesting cross transmission. The presence of van-resistant genes from Enterococcus spp. was not detected. Pets should be considered possible vehicles of transmission and reservoirs for MRS bacteria and veterinary hospitals should be considered high-risk environments for the occurrence and spread of nosocomial infections and resistant bacteria.

Biology of Hand-to-Hand Bacterial Transmission.

Numerous studies have demonstrated that adequate hand hygiene among hospital staff is the best measure to prevent hand-to-hand bacterial transmission. The skin microbiome is conditioned by the individual physiological characteristics and anatomical microenvironments. Furthermore, it is important to separate the autochthonous resident microbiota from the transitory microbiota that we can acquire after interactions with contaminated surfaces. Two players participate in the hand-to-hand bacterial transmission process: the bacteria and the person. The particularities of the bacteria have been extensively studied, identifying some genera or species with higher transmission efficiency, particularly those linked to nosocomial infections and outbreaks. However, the human factor remains unstudied, and intrapersonal particularities in bacterial transmission have not been yet explored. Herein we summarize the current knowledge on hand-to-hand bacterial transmission, as well as unpublished results regarding interindividual and interindividual transmission efficiency differences. We designed a simple in vivo test based on four sequential steps of finger-to-finger contact in the same person artificially inoculated with a precise bacterial inoculum. Individuals can be grouped into one of three observed transmission categories: high, medium, and poor finger-to-finger transmitters. Categorization is relevant to predicting the ultimate success of a human transmission chain, particularly for the poor transmitters, who have the ability to cut the transmission chain. Our model allowed us to analyze transmission rate differences among five bacterial species and clones that cause nosocomial infections, from which we detected that Gram-positive microorganisms were more successfully transmitted than Gram-negative.

Intestinal Immune Dysregulation Driven by Dysbiosis Promotes Barrier Disruption and Bacterial Translocation in Rats With Cirrhosis.

In cirrhosis, intestinal dysbiosis, intestinal barrier impairment, and systemic immune system abnormalities lead to gut bacterial translocation (GBT) and bacterial infection. However, intestinal immune system dysfunction and its contribution to barrier damage are poorly understood. This study correlates immune system dysregulation in the intestines of rats at different stages of CCl4 -induced cirrhosis with barrier function and pathogenic microbiota. The following variables were addressed in the small intestine: intraepithelial lymphocyte (IEL) and lamina propria lymphocyte (LPL) activation status and cytokine production (flow cytometry), cytokine mRNA and protein expression (quantitative real-time PCR and immunofluorescence), microbiota composition of ileum content (16S recombinant DNA massive sequencing), permeability (fecal albumin loss), and epithelial junctions (immunohistochemistry and immunofluorescence). The intestinal mucosa in rats with cirrhosis showed a proinflammatory pattern of immune dysregulation in IELs and LPLs, which featured the expansion of activated lymphocytes, switch to a T helper 1 (Th1) regulatory pattern, and Th17 reduction. In rats with cirrhosis with ascites, this state was associated with epithelial junction protein disruption, fecal albumin loss, and GBT. Direct correlations (P < 0.01) were observed between elevated interferon gamma (IFNγ)-expressing T cytotoxic LPLs and fecal albumin and between inflammatory taxa abundance and IFNγ-producing immune cells in the ileum. Bowel decontamination led to redistributed microbiota composition, reduced proinflammatory activation of mucosal immune cells, normalized fecal albumin levels, and diminished GBT; but there were no modifications in Th17 depletion. Conclusion: The intestinal mucosa of rats with cirrhosis acquires a proinflammatory profile of immune dysregulation that parallels the severity of cirrhosis; this impaired intestinal immune response is driven by gut dysbiosis and leads to disrupted barrier function, promoting GBT.

Optimal Piperacillin-Tazobactam Dosing Strategies against Extended-Spectrum-ß-Lactamase-Producing Enterobacteriaceae.

Piperacillin-tazobactam has been proposed as an alternative to carbapenems for the treatment of infections caused by extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae However, limited understanding of optimal dosing strategies for this combination may curtail its utility. In this study, we correlated various exposures of piperacillin-tazobactam to efficacy, using a modified pharmacokinetic/pharmacodynamic index. Using a clinical Klebsiella pneumoniae isolate expressing CTX-M-15, piperacillin MIC values were determined with increasing tazobactam concentrations and fitted to a sigmoid inhibitory maximum effect (Emax) model. A hollow-fiber infection model (HFIM) was used to evaluate the efficacy of escalating tazobactam dosing with a fixed piperacillin exposure. Simulated drug concentrations from the HFIM were incorporated in the Emax model to determine the percentage of free time above instantaneous MIC (%fT>MICi) associated with each experimental exposure. The target %fT>MICi associated with growth suppression was prospectively validated using an SHV-12-producing isolate of Escherichia coli and 2 other CTX-M-15-producing K. pneumoniae isolates. Based on our reference isolate, piperacillin-tazobactam exposures of %fT>MICi of ≥55.1% were associated with growth suppression. Despite underlying differences, these findings were consistent with prospective observations in 3 other clinical isolates. Our modeling approach can be applied relatively easily in the clinical setting, and it appeared to be robust in predicting the effectiveness of various piperacillin-tazobactam exposures. This modified pharmacokinetic/pharmacodynamic index could be used to characterize response to other ß-lactam/ß-lactamase inhibitor combinations.

Infección de herida por mordedura de gato / Cat bite wound infection

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Prevalence of extended-spectrum beta-lactamase and carbapenemase-producing bloodstream isolates of Klebsiella pneumoniae in a tertiary care hospital.

To improve prescribing of empiric therapy, the local molecular epidemiology of extended-spectrum beta-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPCs) in bloodstream isolates of K. pneumoniae were evaluated. Isolates resistant to third generation cephalosporins were screened phenotypically for ESBLs and carbapenemases, and subsequently confirmed by PCR for the presence of ESBL (blaTEM, blaSHV and blaCTX-M) and carbapenemase (blaKPC, blaVIM, blaNDM and blaOXA-48) genes. Hydrolytic activity (functional gene expression) was quantified using a nitrocefin degradation assay and correlated to ceftazidime or meropenem MIC. Clonality was assessed by repetitive element-based PCR. Beta-lactamases were functionally expressed in 13 isolates (15.5%); 7 (53.8%) harboured blaCTX-M-15 and 6 (46.2%) carried the blaKPC-2 gene. Correlation of hydrolytic activity to MIC yielded a coefficient of 98% for isolates expressing ESBLs alone and 56% for carbapenemase producers. Four unique ESBL-expressing clones and five carbapenem-resistant clones were identified. All 13 resistant isolates were susceptible to ceftazidime/avibactam (MIC ≤ 8/4 mg/L).

Determining ß-lactam exposure threshold to suppress resistance development in Gram-negative bacteria.

Objectives: ß-Lactams are commonly used for nosocomial infections and resistance to these agents among Gram-negative bacteria is increasing rapidly. Optimized dosing is expected to reduce the likelihood of resistance development during antimicrobial therapy, but the target for clinical dose adjustment is not well established. We examined the likelihood that various dosing exposures would suppress resistance development in an in vitro hollow-fibre infection model. Methods: Two strains of Klebsiella pneumoniae and two strains of Pseudomonas aeruginosa (baseline inocula of ∼10 8  cfu/mL) were examined. Various dosing exposures of cefepime, ceftazidime and meropenem were simulated in the hollow-fibre infection model. Serial samples were obtained to ascertain the pharmacokinetic simulations and viable bacterial burden for up to 120 h. Drug concentrations were determined by a validated LC-MS/MS assay and the simulated exposures were expressed as C min /MIC ratios. Resistance development was detected by quantitative culture on drug-supplemented media plates (at 3× the corresponding baseline MIC). The C min /MIC breakpoint threshold to prevent bacterial regrowth was identified by classification and regression tree (CART) analysis. Results: For all strains, the bacterial burden declined initially with the simulated exposures, but regrowth was observed in 9 out of 31 experiments. CART analysis revealed that a C min /MIC ratio ≥3.8 was significantly associated with regrowth prevention (100% versus 44%, P = 0.001). Conclusions: The development of ß-lactam resistance during therapy could be suppressed by an optimized dosing exposure. Validation of the proposed target in a well-designed clinical study is warranted.

New Insights into the Enterococcus faecium and Streptococcus gallolyticus subsp. gallolyticus Host Interaction Mechanisms.

Enterococcus faecium and Streptococcus gallolyticus subsp. gallolyticus (S. gallolyticus) were classically clustered into the Lancefield Group D streptococci and despite their taxonomic reclassification still share a similar genetic content and environment. Both species are considered as opportunistic pathogens. E. faecium is often associated with nosocomial bacteraemia, and S. gallolyticus is sporadically found in endocarditis of colorectal cancer patients. In both cases, the source of infection is commonly endogenous with a translocation process that launches through the intestinal barrier. To get new insights into the pathological processes preceding infection development of both organisms, we used an in vitro model with Caco-2 cells to study and compare the adhesion, invasion and translocation inherent abilities of 6 E. faecium and 4 S. gallolyticus well-characterized isolates. Additionally, biofilm formation on polystyrene, collagen I and IV was also explored. Overall results showed that E. faecium translocated more efficiently than S. gallolyticus, inducing a destabilization of the intestinal monolayer. Isolates Efm106, Efm121 and Efm113 (p < .001 compared to Ef222) exhibited the higher translocation ability and were able to adhere 2-3 times higher than S. gallolyticus isolates. Both species preferred the collagen IV coated surfaces to form biofilm but the S. gallolyticus structures were more compact (p = .01). These results may support a relationship between biofilm formation and vegetation establishment in S. gallolyticus endocarditis, whereas the high translocation ability of E. faecium high-risk clones might partially explain the increasing number of bacteraemia.

Obeticholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats.

BACKGROUND & AIMS: In advanced cirrhosis, gut bacterial translocation is the consequence of intestinal barrier disruption and leads to bacterial infection. Bile acid abnormalities in cirrhosis could play a role in the integrity of the intestinal barrier and the control of microbiota, mainly through the farnesoid X receptor. We investigated the long-term effects of the farnesoid X receptor agonist, obeticholic acid, on gut bacterial translocation, intestinal microbiota composition, barrier integrity and inflammation in rats with CCl4-induced cirrhosis with ascites. METHODS: Cirrhotic rats received a 2-week course of obeticholic acid or vehicle starting once ascites developed. We then determined: bacterial translocation by mesenteric lymph node culture, ileum expression of antimicrobial peptides and tight junction proteins by qPCR, fecal albumin loss, enteric bacterial load and microbiota composition by qPCR and pyrosequencing of ileum mucosa-attached contents, and intestinal inflammation by cytometry of the inflammatory infiltrate. RESULTS: Obeticholic acid reduced bacterial translocation from 78.3% to 33.3% (p<0.01) and upregulated the expression of the farnesoid X receptor-associated gene small heterodimer partner. Treatment improved ileum expression of antimicrobial peptides, angiogenin-1 and alpha-5-defensin, tight junction proteins zonulin-1 and occludin, and reduced fecal albumin loss and liver fibrosis. Enteric bacterial load normalized, and the distinctive mucosal microbiota of cirrhosis was reduced. Gut immune cell infiltration was reduced and inflammatory cytokine and Toll-like receptor 4 expression normalized. CONCLUSIONS: In ascitic cirrhotic rats, obeticholic acid reduces gut bacterial translocation via several complementary mechanisms at the intestinal level. This agent could be used as an alternative to antibiotics to prevent bacterial infection in cirrhosis.

High Prevalence of Co-Infections by Invasive and Non-Invasive Chlamydia trachomatis Genotypes during the Lymphogranuloma Venereum Outbreak in Spain.

The evolution of Chlamydia trachomatis is mainly driven by recombination events. This fact can be fuelled by the coincidence in several European regions of the high prevalence of non-invasive urogenital genotypes and lymphogranuloma venereum (LGV) outbreaks. This scenario could modify the local epidemiology and favor the selection of new C. trachomatis variants. Quantifying the prevalence of co-infection could help to predict the potential risk in the selection of new variants with unpredictable results in pathogenesis or transmissibility. In the 2009-2013 period, 287 clinical samples with demonstrated presence of C. trachomatis were selected. They were divided in two groups. The first group was constituted by 137 samples with C. trachomatis of the LGV genotypes, and the second by the remaining 150 samples in which the presence of LGV genotypes was previously excluded. They were analyzed to detect the simultaneous presence of non-LGV genotypes based on pmpH and ompA genes. In the first group, co-infections were detected in 10.9% of the cases whereas in the second group the prevalence was 14.6%, which is the highest percentage ever described among European countries. Moreover, bioinformatic analyses suggested the presence among men who have sex with men of a pmpH-recombinant variant, similar to strains described in Seattle in 2002. This variant was the result of genetic exchange between genotypes belonging to LGV and members of G-genotype. Sequencing of other genes, phylogenetically related to pathotype, confirmed that the putative recombinant found in Madrid could have a common origin with the strains described in Seattle. Countries with a high prevalence of co-infections and high migration flows should enhance surveillance programs in at least their vulnerable population.

Individual variability in finger-to-finger transmission efficiency of Enterococcus faecium clones.

A fingertip-to-fingertip intraindividual transmission experiment was carried out in 30 healthy volunteers, using four MLST-typed Enterococcus faecium clones. Overall results showed an adequate fit goodness to a theoretical exponential model, whereas four volunteers (13%) exhibited a significantly higher finger-to-finger bacterial transmission efficiency. This observation might have deep consequences in nosocomial epidemiology.

Emergence and long-lasting persistence of linezolid-resistant Enterococcus faecium-ST117 in an oncohematologic patient after a nine-day course of linezolid.

We report the emergence and long-lasting persistence of linezolid resistance in an ampicillin-resistant Enterococcus faecium strain in the intestine of a neutropenic oncohematologic patient receiving chemotherapy. The patient was first colonized by an epidemic ampicillin-resistant E. faecium (ARE)-ST117 clustering into lineage 78. This clone exhibited resistance to levofloxacin, erythromycin and high-level resistance to streptomycin and gentamicin. After receiving treatment with several broad spectrum antibiotics for febrile neutropenia, a 9-day course of oral linezolid was administered once the patient developed bacteraemia by the same ARE colonizing clone. Linezolid-resistant ARE was detected 17 days later in the follow-up fecal samples and persisted 41 days after suppression of linezolid therapy. Resistance to linezolid was associated with G2576T transversion in the 23S rRNA and the presence of cfr gene was not detected. The persistence of G2576T-ARE strains, especially in oncohematologic patients with injured intestinal membranes, could increase the risk of bacteraemia.

Mechanisms of linezolid resistance among Staphylococci in a tertiary hospital.

The mechanisms of linezolid resistance among 86 staphylococcal isolates from two intensive care units were investigated. The most frequent was the G2576T mutation in the 23S rRNA (82%). The cfr gene was found in 17% of the isolates, seven S. aureus and eight S. epidermidis isolates. Four of the S. epidermidis isolates had the G2576T mutation and the cfr gene. In four S. haemolyticus isolates, the mechanism could not be identified.