Enterobacterales carrying chromosomal AmpC ß-lactamases in Europe (EuESCPM): Epidemiology and antimicrobial resistance burden from a cohort of 27 hospitals, 2020-2022.

INTRODUCTION: The ESCPM group (Enterobacter species including Klebsiella aerogenes - formerly Enterobacter aerogenes, Serratia species, Citrobacter freundii complex, Providencia species and Morganella morganii) has not yet been incorporated into systematic surveillance programs. METHODS: We conducted a multicentre retrospective observational study analysing all ESCPM strains isolated from blood cultures in 27 European hospitals over a 3-year period (2020-2022). Diagnostic approach, epidemiology, and antimicrobial susceptibility were investigated. RESULTS: Our study comprised 6,774 ESCPM isolates. MALDI-TOF coupled to mass spectrometry was the predominant technique for bacterial identification. Susceptibility to new ß-lactam/ß-lactamase inhibitor combinations and confirmation of AmpC overproduction were routinely tested in 33.3% and 29.6% of the centres, respectively. The most prevalent species were E. cloacae complex (44.8%) and S. marcescens (22.7%). Overall, third-generation cephalosporins (3GC), combined third- and fourth-generation cephalosporins (3GC + 4GC) and carbapenems resistance phenotypes were observed in 15.7%, 4.6%, and 9.5% of the isolates, respectively. AmpC overproduction was the most prevalent resistance mechanism detected (15.8%). Among carbapenemase-producers, carbapenemase type was provided in 44.4% of the isolates, VIM- (22.9%) and OXA-48-enzyme (16%) being the most frequently detected. E. cloacae complex, K. aerogenes and Providencia species exhibited the most notable cumulative antimicrobial resistance profiles, with the former displaying 3GC, combined 3GC + 4GC and carbapenems resistance phenotypes in 15.2%, 7.4%, and 12.8% of the isolates, respectively. K. aerogenes showed the highest rate of both 3GC resistant phenotype (29.8%) and AmpC overproduction (32.1%), while Providencia species those of both carbapenems resistance phenotype (42.7%) and carbapenemase production (29.4%). ESCPM isolates exhibiting both 3GC and combined 3GC + 4GC resistance phenotypes displayed high susceptibility to ceftazidime/avibactam (98.2% and 95.7%, respectively) and colistin (90.3% and 90.7%, respectively). Colistin emerged as the most active drug against ESCPM species (except those intrinsically resistant) displaying both carbapenems resistance phenotype (85.8%) and carbapenemase production (97.8%). CONCLUSIONS: This study presented a current analysis of ESCPM species epidemiology in Europe, providing insights to inform current antibiotic treatments and guide strategies for antimicrobial stewardship and diagnostics.

COVID-19 Biomarkers Comparison: Children, Adults and Elders.

Background and Objectives: this study aimed to research links between C-reactive protein (CRP), lactate dehydrogenase (LDH), creatinekinase (CK), 25-OH vitamin D (25-OHD), ferritin (FER), high-density lipoprotein cholesterol (HDL)cholesterol and clinical severity in patients from the western part of Romania, and compare their potential use as biomarkers for intensive care units (ICU) admission and death in children, adults and elders. Materials and Methods: this study is a retrospective cohort study, performed on patients positively diagnosed with COVID-19. Available CRP, LDH, CK 25-OH vitamin D, ferritin, HDL cholesterol and clinical severity were recorded. The following were assessed: median group differences, association, correlation and receiver operating characteristic. Results: 381 children, 614 adults and 381 elders were studied between 1 March 2021 and 1 March 2022. Most children and adults presented mild symptomatology (53.28%, 35.02%, respectively), while most elders presented severe symptomatology (30.04%). ICU admission was 3.67% for children, 13.19% for adults and 46.09% for elders, while mortality was 0.79% for children, 8.63% for adults and 25.1% for elders. With the exception of CK, all other biomarkers showed some significant associations with clinical severity, ICU admission and death. Conclusions: CRP, LDH, 25-OH vitamin D, ferritin and HDL are important biomarkers for COVID-19 positive patients, especially in the pediatric population, while CK was mostly within normal ranges.

The Challenges of The Diagnostic and Therapeutic Approach of Patients with Infectious Pathology in Emergency Medicine.

The emergency department (ED) represents an important setting for addressing inappropriate antimicrobial prescribing practices because of the time constraints and the duration of microbiological diagnosis. The purpose of this study is to evaluate the etiology and antimicrobial resistance (AMR) pattern of the community-acquired pathogens, as well as the epidemiological characteristics of patients admitted through the ED, in order to guide appropriate antibiotic therapy. METHODS: A retrospective observational study was performed on 657 patients, from whom clinical samples (urine, purulent secretions, blood cultures, etc.) were collected for microbiological diagnosis in the first 3 days after presentation in the ED. The identification of pathogens and the antimicrobial susceptibility testing with minimum inhibitory concentration determination were carried out according to the laboratory protocols. RESULTS: From the 767 biological samples analyzed, 903 microbial isolates were identified. E. coli was most frequently isolated (24.25%), followed by Klebsiella spp., S. aureus (SA), and non-fermentative Gram-negative bacilli. E. coli strains maintained their natural susceptibility to most antibiotics tested. In the case of Pseudomonas spp. and Acinetobacter spp., increased rates of AMR were identified. Also, 32.3% of SA strains were community-acquired MRSA. CONCLUSIONS: The introduction of rapid microbiological diagnostic methods in emergency medicine is imperative in order to timely identify AMR strains and improve therapeutic protocols.

Bacterial and Fungal Superinfections in COVID-19 Patients Hospitalized in an Intensive Care Unit from Timișoara, Romania.

Purpose: Critically ill patients hospitalized in the intensive care unit (ICU) have an increased infection risk. The aim of this study was to determine the bacterial and fungal superinfections rate in Coronavirus disease 2019 (COVID-19) patients stationed in the ICU, identify risk factors associated with their development and to determine whether superinfection plays a role in patients' outcome in this population. Patients and Methods: In this retrospective, non-interventional, single centre, cohort study, medical records of 302 consecutive patients with SARS-COV-2 pneumonia admitted into the COVID-19 ICU of the largest university hospital from Western Romania between October 2020 and May 2021, were reviewed, of whom 236 patients met the inclusion criteria. Results: One hundred and nineteen patients developed a superinfection ≥48 h after being admitted to the hospital. Superinfection rate in the ICU was 50.42%. Coagulase-negative Staphylococci (CoNS) and Enterococcus spp. were predominantly isolated from blood cultures, while Acinetobacter baumannii, Staphylococcus aureus and Candida spp. from tracheobronchial aspirates. Significant independent risk factors regarding bacterial/fungal superinfection in COVID-19 patients were obtained for the following variables: number of days of central venous catheter (HR = 1.13 [1.07-1.20], p < 0.001) and prior administration of corticosteroids (HR = 2.80 [1.33-5.93], p = 0.007). Four independent predictive risk factors were associated with unfavorable outcome: age (HR = 1.07 [95% CI 1.03-1.12], p = 0.001); Carmeli Score (HR = 6.09 [1.18-31.50], p = 0.031); body mass index (HR = 1.11 [1.02-1.21], p = 0.011) and the presence of a central venous catheter (HR = 6.49 [1.93-21.89], p = 0.003). Conclusion: The superinfection rate in COVID-19 patients was high in this study group. Exogenous risk factors were associated with superinfection more than endogenous factors. Only a small percentage of uninfected COVID-19 patients were not prescribed antibiotics during their hospitalization, raising serious concerns regarding the judicious prescribing of antibiotics in viral infections.

Encrusted Uretero-Pyelitis Caused by Corynebacterium urealyticum: Case Report and Literature Review.

We report the case of a 70-year-old female patient with solitary functioning left kidney and encrusted uretero-pyelitis caused by Corynebacterium urealyticum, which was treated by antibiotic therapy and oral acidification with L-methionine. We review the literature for similarly reported cases.

Self-reported antibiotic stewardship and infection control measures from 57 intensive care units: An international ID-IRI survey.

We explored the self-reported antibiotic stewardship (AS), and infection prevention and control (IPC) activities in intensive care units (ICUs) of different income settings. A cross-sectional study was conducted using an online questionnaire to collect data about IPC and AS measures in participating ICUs. The study participants were Infectious Diseases-International Research Initiative (IDI-IR) members, committed as per their institutional agreement form. We analyzed responses from 57 ICUs in 24 countries (Lower-middle income (LMI), n = 13; Upper-middle income (UMI), n = 33; High-income (HI), n = 11). This represented (~5%) of centers represented in the ID-IRI. Surveillance programs were implemented in (76.9%-90.9%) of ICUs with fewer contact precaution measures in LMI ones (p = 0.02); (LMI:69.2%, UMI:97%, HI:100%). Participation in regional antimicrobial resistance programs was more significantly applied in HI (p = 0.02) (LMI:38.4%,UMI:81.8%,HI:72.2%). AS programs are implemented in 77.2% of institutions with AS champions in 66.7%. Infectious diseases physicians and microbiologists are members of many AS teams (59%&50%) respectively. Unqualified healthcare professionals(42.1%), and deficient incentives(28.1%) are the main barriers to implementing AS. We underscore the existing differences in IPC and AS programs' implementation, team composition, and faced barriers. Continuous collaboration and sharing best practices on APM is needed. The role of regional and international organizations should be encouraged. Global support for capacity building of healthcare practitioners is warranted.

The Successful Recovery of a Critically Ill COVID-19 Patient, Following the Combination of Therapeutic Plasma Exchange and Convalescent Plasma Transfusion: A Case Report.

With an intricate symptom pattern involving a dysregulated host response to infection, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause severe inflammation and cytokine storms, acute respiratory distress syndrome, coagulopathy, multi-organ failure, and finally death. The uniqueness of this case report lies in the nature of the therapeutic intervention performed. While numerous studies are available on both the use of therapeutic plasma exchange in coronavirus disease 2019 (COVID-19) patients and convalescent plasma transfusion as separate treatment methods, there is very little information regarding the combination of these procedures. We present the case of a 52-year-old male, unvaccinated for COVID-19, who tested positive on reverse transcriptase polymerase chain reaction for SARS-CoV-2 for the first time and presented in the emergency room with fever, chills, severe cough, tachypnea, tachycardia, and dyspnea that started two days before presentation. Upon rapid assessment, the patient showed signs of acute respiratory failure, so it was decided to transfer the patient to the intensive care unit, COVID-19 ward, after preliminary radiological examination. For the next 24 days, the patient was stationed in the intensive care unit, where he was closely monitored and treated. Invasive mechanical ventilation was required following the initial worsening of his respiratory status. We performed therapeutic plasma exchange on the first day of his stay in the intensive care unit, and immediately after the procedure, the patient was transfused with 500 mL of convalescent plasma from healthy donors. The patient's condition improved over the next few days, which led to the cessation of mechanical ventilation and, after treating the superinfection, the patient was discharged home, making a full recovery. The early initiation of therapeutic plasma exchange followed by transfusion of convalescent plasma in severe and critical forms of COVID-19 may reduce the risk of the progression of the disease and ultimately reduce the risk of negative outcomes in a selected group of patients.

Severe Thrombocytopenic Purpura Associated with COVID-19 in a Pediatric Patient.

Purpose: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to cause a diverse spectrum of clinical manifestations ranging from mild, flu-like symptoms to severe progressive pneumonia, acute respiratory distress syndrome with or without other extrapulmonary impairment. Hematological changes such as lymphopenia, neutrophilia, and anemia as the disease progresses, are frequently found in COVID-19. Thrombocytopenia may be drug-induced or can occur secondary to sepsis, disseminated intravascular coagulation or bone marrow suppression. Immune thrombocytopenic purpura (ITP) is frequently observed in children aged 2-5 years and in 60% of cases may proceed an upper respiratory tract infection. The present paper aimed to raise awareness of ITP as a possible pediatric presentation of coronavirus disease. Patients and Methods: We present the case of previously healthy, eight-year-old female patient, who developed an immune thrombocytopenia flare, also known as immune thrombocytopenic purpura (ITP), in the context of COVID-19, with diffuse petechiae and ecchymosis on her body, face and oral mucosa, and a nadir platelet count of 0×103/µL. Results: Platelet count recovery was observed after seven days of combined treatment with intravenous immunoglobulin (IVIG) and corticosteroids. Conclusion: The growing body of literature regarding the clinical and laboratory manifestations of COVID-19 infection in children, has reported thrombocytopenia in relation to unfavorable disease progression or multisystem inflammatory syndrome (MIS-C). Clinicians must be aware that ITP may appear both in mild and severe COVID-19, at any time during its course, and can be associated with a higher bleeding risk, thus its diagnostic may be critical.

Current State of Knowledge Regarding WHO Critical Priority Pathogens: Mechanisms of Resistance and Proposed Solutions through Candidates Such as Essential Oils.

The rise of multidrug-resistant (MDR) pathogens has become a global health threat and an economic burden in providing adequate and effective treatment for many infections. This large-scale concern has emerged mainly due to mishandling of antibiotics (ABs) and has resulted in the rapid expansion of antimicrobial resistance (AMR). Nowadays, there is an urgent need for more potent, non-toxic and effective antimicrobial agents against MDR strains. In this regard, clinicians, pharmacists, microbiologists and the entire scientific community are encouraged to find alternative solutions in treating infectious diseases cause by these strains. In its "10 global issues to track in 2021", the World Health Organization (WHO) has made fighting drug resistance a priority. It has also issued a list of bacteria that are in urgent need for new ABs. Despite all available resources, researchers are unable to keep the pace of finding novel ABs in the face of emerging MDR strains. Traditional methods are increasingly becoming ineffective, so new approaches need to be considered. In this regard, the general tendency of turning towards natural alternatives has reinforced the interest in essential oils (EOs) as potent antimicrobial agents. Our present article aims to first review the main pathogens classified by WHO as critical in terms of current AMR. The next objective is to summarize the most important and up-to-date aspects of resistance mechanisms to classical antibiotic therapy and to compare them with the latest findings regarding the efficacy of alternative essential oil therapy.

Listeria Monocytogenes Meningitis in an Immunocompetent Patient.

Listeria monocytogenes (LM), one of the most important foodborne pathogens, is an intracellular bacterium found in food and the environment. It causes listeriosis, a potentially severe disease, particularly for pregnant women, the elderly and immunocompromised patients, but in rare cases, it can cause invasive disease in immunocompetent adults and children. Community-acquired bacterial meningitis caused by LM is rare and difficult to diagnose. It carries a high mortality rate; therefore, it is essential to start appropriate antibiotic treatment as soon as possible. The first case of LM meningitis identified in our hospital over the last 10 years is that of a previously healthy 45-year-old man who presented in the emergency department with a 4-day history of diplopia, left eye medial deviation and left palpebral ptosis, with no history of fever, headache or gastrointestinal symptoms. Because of the atypical symptoms, a suspicion of meningitis vs cerebral aneurysm was raised during the admission process. The patient was diagnosed with LM meningitis and recovered fully after appropriate antibiotic treatment. The purpose of this article is to emphasise the possibility of LM invasive disease (in this case meningitis) occurring in previously healthy individuals and to raise awareness about the need for LM to be considered in the differential diagnosis of atypical presentations.

Association of Proteus mirabilis and Providencia stuartii Infections with Diabetes.

Background and Objectives: Proteus and Providencia are related genera of opportunistic pathogens belonging to the Morganellaceae family, often a cause of infections in the immunocompromised hosts, such as diabetic patients. Their clinical significance has increased due to their intrinsic resistance to polymyxins, which is often associated with acquired resistance mechanisms. In this study we evaluated the infections caused by Proteus mirabilis and Providencia stuartii in two groups of patients, with diabetes (group 1) and without diabetes (group 2) admitted to the intensive care unit and surgical wards. The infections were investigated in terms of infection type, risk factors, clinical course, predictive factors for unfavourable outcomes and antibiotic resistance profile. Materials and Methods: An observational, retrospective, cross-sectional study was conducted, comprising all patients infected with these pathogens. Bacterial identification and antibiotic sensitivity testing were performed using the Vitek2C automated system. Results: Comparison of the two groups showed that the statistically significant common infectious risk factors were found less frequently among diabetic patients when compared with non-diabetic patients, and that antimicrobial resistance was significantly lower in the diabetic patient group. However, survival rates did not differ between the two groups, drawing attention to the implications of diabetes as comorbidity. Additionally, with regard to the antibiotic resistance profile, 38.89% of P. stuartii strains isolated from diabetic patients belonged to the difficult-to-treat (DTR) phenotype, contributing to the severity of these infections compared with those caused by P. mirabilis, of which 32% were wild type strains and 0% were DTR phenotype. The DTR/extended spectrum beta-lactamase producing P. stuartii isolates more than doubled the risk of mortality, while the presence of nasogastric nutrition tripled the risk. Conclusions: P. stuartii infections that occurred in diabetic patients proved to be more difficult to treat, the majority of them being healthcare-associated bacteremias.

Therapeutic plasma exchange followed by convalescent plasma transfusion in severe and critically ill COVID-19 patients: A single centre non-randomized controlled trial.

Therapeutic plasma exchange (TPE) has been proposed as a rescue therapy in critically ill COVID-19 patients. The aim of the present study was to determine whether combining TPE with convalescent plasma (CVP) transfusion early in the intensive care unit (ICU) stay improves survival among this heterogeneous population. The primary endpoint was survival at 30 days. Secondary endpoints included assessing the evolution of biomarkers, such as the partial pressure of arterial oxygen to fractional inspired oxygen ratio, and C reactive protein (CRP), lactate dehydrogenase (LDH) and ferritin levels at the 7-day follow-up. This single centre, prospective, non-randomized controlled trial was conducted in an 8-bed COVID-19 ICU and included patients with severe COVID-19 pneumonia requiring intensive care treatment. A total of 19 patients were treated by performing TPE followed by CVP transfusion, in addition to standard treatment, while for another 19 patients, only standard treatment according to hospital protocols was used. TPE was initiated during the first 24 h after ICU admission, followed immediately by transfusion of CVP. Survival at 30 days was 47.37% in the TPE CVP group and 26.32% in the control group (P=0.002). Patients in the TPE CVP group also showed better oxygenation and a reduction in inflammation, with decreased CRP, LDH and ferritin levels compared with those in the control group. Overall, the study indicated that early initiation of TPE followed by CVP transfusion may be a valid rescue therapy in severe and critically ill COVID-19 patients, with a statistically significant survival benefit, improved oxygenation and a reduction in inflammatory markers. The trial was registered in the ClinicalTrials.gov database (trial registration number: NCT04973488) on July 22, 2021 (retrospectively registered).

Association between ribotype and clinical form of enterocolitis with C. difficile in Western Romania: A 2-year study.

During the last decade, several changes in the epidemiology, clinical symptoms, and evolution of Clostridium difficile infection (CDI) have been reported. The number of diagnosed cases has increased, especially in individuals over 60 years of age. There has also been an increase in the share of severe forms of the disease, the number of patients with recurrent infections and the lethality caused by this condition. The aim of the present study was to determine the incidence of CDI over the past few years; to monitor the bacterial toxin by ribotyping; to observe the migration of circulating toxins; to correlate the ribotyping with the clinical form of the disease; and to correlate the treatment with the ribotyping and the clinical form. Therefore, we performed an observational retrospective study regarding the incidence of CDI at 'Victor Babeș' Hospital of Infectious Diseases and Pneumophtisiology of Timișoara, between January 2016 and December 2017. The results revealed that 210 patients were hospitalized with a diagnosis of acute enterocolitis with C. difficile. All patients tested showed C. difficile toxin A/B positivity. In 28 cases, the ribotyping was positive with binary toxin and hypervirulent strain 027. The tested patients were discharged or transferred from various medical/surgical clinics. In the study, we also ascertained the correlation between the clinical form of the disease and comorbidities and pre-hospital treatments as risk factors in the occurrence of the infection. In accordance with the rise of CDI during the last decade, it was possible to observe the increase in the number of infectious recurrences. From this point of view, highlighting of the ribotype in this pathology becomes a primary aspect both by increasing the success rate in curing the disease (with a reduction in the risk of recurrence) and by targeted and non-empirical administration of the antibiotic.

Clostridium Difficile and COVID-19: General Data, Ribotype, Clinical Form, Treatment-Our Experience from the Largest Infectious Diseases Hospital in Western Romania.

Background and Objectives: In Coronavirus Disease 2019 (COVID-19), which is caused by the infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the clinical manifestations are primarily related to the pulmonary system. Under 10% of cases also develop gastrointestinal events such as diarrhea, nausea, vomiting and abdominal pain. Materials and Methods: We conducted an observational, retrospective study in the Infectious Diseases Clinic of "Victor Babes" Hospital, Timis County, in order to assess the incidence, outcome and risk factors for clostridium difficile infection (CDI) in COVID-19 patients. Results: Out of 2065 COVID-19 cases, hospitalized between 1st September 2020 and 30th April 2021, 40 cases of CDI were identified with 32 cases of hospital-onset of CDI and eight cases of community-onset and healthcare-associated CDI. By randomization, polymerase chain reaction ribotyping of Clostridium Difficile was performed in six cases. All the randomized cases tested positive for ribotype 027. The percentage of cases recovered with complications at discharge was higher among COVID-19 patients and CDI (p = 0.001). The in-hospital stay, 36 days versus 28 days, was longer among COVID-19 patients and CDI (p = 0.01). The presence of previous hospitalization (p = 0.004) and administration of antibiotics during the hospital stay, increased the risk of CDI among COVID-19 patients. The mean adjusted CCI at admission was lower among controls (p = 0.01). In two cases, exitus was strictly CDI-related, with one case positive for 027 ribotype. Conclusions: CDI has complicated the outcome of COVID-19 patients, especially for those with comorbidities or previously exposed to the healthcare system. In the face of the COVID-19 pandemic and the widespread, extensive use of antibiotics, clinicians should remain vigilant for possible CDI and SARS-CoV-2 co-infection.

Profiles of multidrug-resistant organisms among patients with bacteremia in intensive care units: an international ID-IRI survey.

Evaluating trends in antibiotic resistance is a requisite. The study aimed to analyze the profile of multidrug-resistant organisms (MDROs) among hospitalized patients with bacteremia in intensive care units (ICUs) in a large geographical area. This is a 1-month cross-sectional survey for blood-borne pathogens in 57 ICUs from 24 countries with different income levels: lower-middle-income (LMI), upper-middle-income (UMI), and high-income (HI) countries. Multidrug-resistant (MDR), extensively drug-resistant (XDR), or pan-drug-resistant isolates were searched. Logistic regression analysis determined resistance predictors among MDROs. Community-acquired infections were comparable to hospital-acquired infections particularly in LMI (94/202; 46.5% vs 108/202; 53.5%). Although MDR (65.1%; 502/771) and XDR (4.9%; 38/771) were common, no pan-drug-resistant isolate was recovered. In total, 32.1% of MDR were Klebsiella pneumoniae, and 55.3% of XDR were Acinetobacter baumannii. The highest MDR and XDR rates were in UMI and LMI, respectively, with no XDR revealed from HI. Predictors of MDR acquisition were male gender (OR, 12.11; 95% CI, 3.025-15.585) and the hospital-acquired origin of bacteremia (OR, 2.643; 95%CI, 1.462-3.894), and XDR acquisition was due to bacteremia in UMI (OR, 3.344; 95%CI, 1.189-5.626) and admission to medical-surgical ICUs (OR, 1.481; 95% CI, 1.076-2.037). We confirm the urgent need to expand stewardship activities to community settings especially in LMI, with more paid attention to the drugs with a higher potential for resistance. Empowering microbiology laboratories and reports to direct prescribing decisions should be prioritized. Supporting stewardship in ICUs, the mixed medical-surgical ones in particular, is warranted.

Detection of Extended-Spectrum ß-Lactamase and Carbapenemase Activity in Gram-Negative Bacilli Using Liquid Chromatography - Tandem Mass Spectrometry.

PURPOSE: Several mass spectrometry-based methods for antimicrobial sensitivity testing have been described in recent years. They offer an alternative to commercially available testing systems which were considered to have disadvantages in terms of cost- and time-efficiency. The aim of this study was to develop an LC-MS/MS-based antibiotic hydrolysis assay for evaluating antimicrobial resistance (AMR) of Gram-negative bacteria. MATERIALS AND METHODS: Four species of Gram-negative bacilli (Klebsiella pneumoniae, Escherichia coli, Providencia stuartii and Acinetobacter baumannii) were tested against six antibiotics from three different classes: ampicillin, meropenem, imipenem, ceftazidime, ceftriaxone and cefepime. Bacterial suspensions from each species were incubated with a mixture of the six antibiotics. Any remaining antibiotic following incubation was measured using LC-MS/MS. The results were interpreted using measurements obtained for an E. coli strain sensitive to all antibiotics and expressed as percentage of hydrolyzed antibiotic. These were subsequently compared to commercially-available system for the bacteria identification and susceptibility testing. RESULTS: Overall, LC-MS/MS assay and commercial antimicrobial susceptibility platform results showed good agreement in terms of an organism being resistant/sensitive to an antibiotic. The time required to complete the LC-MS/MS-based hydrolysis test was under 5 h, significantly shorter that commercially available susceptibility testing platforms. CONCLUSION: By using a sensitive strain for results interpretation and simultaneous use of multiple antibiotics, the proposed protocol offers improved robustness and multiplexing over previously described methods for antibiotic sensitivity testing. Nevertheless, further research is needed before routine assimilation of the method, especially for strains with intermediate resistance.

Urinary Microbiota-Are We Ready for Prime Time? A Literature Review of Study Methods' Critical Steps in Avoiding Contamination and Minimizing Biased Results.

Within the last few years, there have been an increased number of clinical studies involving urinary microbiota. Low-biomass microbiome sequencing (e.g., urine, lung, placenta, blood) is easily biased by contamination or cross-contamination. So far, a few critical steps, from sampling urine to processing and analyzing, have been described (e.g., urine collection modality, sample volume size, snap freezing, negative controls usage, laboratory risks for contamination assessment, contamination of negative results reporting, exploration and discussion of the impact of contamination for the final results, etc.) We performed a literature search (Pubmed, Scopus and Embase) and reviewed the published articles related to urinary microbiome, evaluating how the aforementioned critical steps to obtain unbiased, reliable results have been taken or have been reported. We identified different urinary microbiome evaluation protocols, with non-homogenous reporting systems, which can make gathering results into consistent data for similar topics difficult and further burden the already so complex emerging field of urinary microbiome. We concluded that to ease the progress in this field, a joint approach from researchers, authors and publishers would be necessary in order to create mandatory reporting systems which would allow to recognize pitfalls and avoid compromising a promising field of research.

Distribution of NDM1 Carbapenemase-Producing Proteeae Strains on High-Risk Hospital Wards.

BACKGROUND: Carbapenem-resistant Proteeae (CRP) is a group of multidrug-resistant (MDR) microorganisms that raise special treatment problems due to their intrinsic resistance to colistin. In this study, our aim is to provide a phenotypic and molecular characterization of the carbapenemases secreted by CRP strains isolated from inpatients from an intensive care unit (ICU) and surgical wards, as well as the identification of the risk factors involved in their acquisition. METHODS: An observational, cross-sectional study was performed which included all Proteeae strains isolated in samples from inpatients on high-risk wards of the largest university hospital in Western Romania, from July 2017 to April 2019. Meropenem-resistant strains (N=65) with MIC ≥16 µg/mL were subjected to a singleplex PCR assay for the detection of blaNDM, blaVIM and blaCTX-M genes. The analysis of risk factors was performed by logistic regression. RESULTS: Out of 8317 samples that were processed, 400 Proteeae strains were isolated: 64% belonging to the genus Proteus, 26.75% to the genus Providencia and 9.25% to the genus Morganella. Most CRP strains (N=56) were of MBL type, and 55 had the blaNDM gene as the prevalent gene substrate. P. stuartii was the main species that provided the circulating MDR strains. Most CRP strains came from patients admitted to ICU, being isolated mainly from bronchial aspirates and blood cultures. Multivariate analysis revealed 3 independent risk factors - mechanical ventilation>96h (HR: 40.51 [13.65-120.25], p <0.001), tracheostomy (HR: 2.65 [1.14-6.17], p = 0.024) and prolonged antibiotic therapy (HR: 1.01 [1.00-1.02], p = 0.03). CONCLUSION: There is a significant increase in the incidence of CR P. stuartii strains, the MBL-blaNDM type being predominant. These strains presented various other resistance mechanisms, being often extremely difficult to treat and led to an excess of lethality of 27.16%.

Genetic Features of mcr-1 Mediated Colistin Resistance in CMY-2-Producing Escherichia coli From Romanian Poultry.

Colistin is a last resort antibiotic used for the treatment of human infections associated with carbapenemase-producing Enterobacteriales. Here, we evaluated the occurrence of mcr-1 and -2 plasmid-mediated colistin resistance in colistin and/or carbapenem resistant human clinical Enterobacteriales and other gram-negative bacteria (n = 543) as well as third generation cephalosporin-resistant (3GCR) Escherichia coli isolates from poultry abattoir workers (n = 15) and poultry fecal samples (n = 92) collected from two geographically separate abattoirs in Romania. which revealed that mcr-1 was present within four sequence types (STs): ST744 (n = 7), ST57 (n = 7), ST156 (n = 2), and ST10 (n = 1). Within STs, serotypes were conserved and, notably, all except one of the mcr-1-positive isolates were found to exhibit fluoroquinolone-resistance (FQR) associated SNPs in both gyrA and parC. While there were variations in genotypes, all isolates belonging to ST744, ST57, and ST156 were rich in resistance determinants, carrying aminoglycoside-modifying enzymes genes, sulfonamide resistance gene bla TEM- 1 as well as bla CMY- 2 AmpC ß-lactamase resistance genes. They also exhibited high similarity in carriage of virulence genes; ST10, however, only carried the mcr-1 gene. Whole genome sequencing (WGS) analysis also revealed that although the mcr-1 gene was identified in a diverse population of E. coli, two STs (ST57 and ST744) predominated and interestingly, were found in isolates across both abattoirs providing evidence for clonal transmission. Also, two main genomic contexts of mcr-1 isolates were revealed with all ST57 isolates harboring the mcr-1 gene between two copies of ISApl1 (or the Tn6330 transposon) whilst a common mcr-1 containing scaffold, highly similar to IncX type mcr-1-bearing plasmids (pWI2-mcr, Accession number: LT838201), was present among mcr-1 isolates of varying phylogenetic backgrounds (ST10, ST744 and ST156). The high prevalence of the mcr-1 gene in poultry E. coli isolates with co-resistance to cephalosporins and quinolones, in a country where antimicrobial use in food production species is poorly regulated, is concerning and the findings from this study should lead to better surveillance of antimicrobial resistance (AMR) in food-production animals in Romania.

Evaluation of essential oil obtained from Mentha×piperita L. against multidrug-resistant strains.

BACKGROUND: Bacterial multidrug resistance currently poses an increasingly serious threat, with important clinical consequences regarding treatment options. In 2017, the WHO released a global list of resistant bacteria, identifying multidrug-resistant (MDR) Gram-negative bacteria such as carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa or Acinetobacter baumannii, extended-spectrum cephalosporin-resistant Enterobacteriaceae as critical priorities for developing new strategies of treatment. PURPOSE: The novelty presented in this study refers to the evaluation of the volatile oil obtained from the leaves of Mentha×piperita L., on MDR strains from hospitalized patients. MATERIAL AND METHODS: The essential oil was extracted by steam distillation and tested on six reference bacterial strains and also on the MDR strains collected from patients of the "Pius Brînzeu" Emergency Clinical County Hospital Timișoara. The in vitro antibacterial activity was evaluated by agar disk diffusion method and microdilution method. RESULTS: Testing the antibacterial activity of peppermint oil on both reference strains and isolated MDR strains from hospitalized patients demonstrated its bactericidal effect. Minimum inhibitory concentration (MIC) was lower (20 mg/mL) for Staphylococcus aureus, Escherichia coli and Proteus mirabilis and higher (40 mg/mL) for Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii strains. Minimum bactericidal concentration (MBC) was equal to MIC, with the exception of Pseudomonas aeruginosa strains, where MBC was the double of MIC. CONCLUSION: The present study highlights the bactericidal activity of Mentha×piperita L. essential oil on all tested MDR or extensively drug-resistant Gram-positive and Gram-negative strains of Staphylococcus aureus, Escherichia coli, Klebsiellapneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Acinetobacter baumannii. This oil may be a therapeutic option in the near future for many infectious diseases produced by MDR bacteria.