Evaluation of a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage and its application in rapid ultrasensitive identification of Acinetobacter baumannii.

BACKGROUND: Rapid phage enumeration/quantitation and viable bacteria determination is critical for phage application and treatment of infectious patients caused by the pathogenic bacteria. METHODS: In the current study, a direct phage DNA detection-based Taqman qPCR methodology for quantification of phage P53 and rapid ultrasensitive identification of Acinetobacter baumannii (A. baumannii) was evaluated. RESULTS: The assay was capable of quantifying P53 phage DNA without DNA extraction and the detection limit of the assay was 550 PFU/mL. The agreement bias between the quantitative results of three different phage concentrations in this assay and double agar overlay plaque assay were under 3.38%. Through the built detection system, down to 1 log CFU/mL of viable A. baumannii can be detected within 4 h in A. baumannii spiked swab and bronchoalveolar lavage fluid samples. Compared with the Taqman qPCR that targets the conserved sequence of A. baumannii, the sensitivity of the assay built in this study could increase four orders of magnitude. CONCLUSIONS: The methodology offers a valid alternative for enumeration of freshly prepared phage solution and diagnosis of bacterial infection caused by A. baumannii or other bacterial infection in complicated samples through switching to phages against other bacteria. Furthermore, the assay could offer drug adjustment strategy timely owing to the detection of bacteria vitality.

False Laboratory Test Result Through Colistin Interference in an Intensive Care Patient: Case Report.

BACKGROUND: In blood samples taken for testing purposes during drug infusion in the intensive care unit, there is a risk of interference due to drug-reactive interaction during the analysis. CASE REPORT: A 19-year-old female patient had undergone surgery for intracranial astrocytoma, 12 years ago. Acinetobacter baumannii was found in the blood culture and deep tracheal aspiration fluid of the patient who had a fever (39.2 °C) with a body temperature during the follow-up. The patient was started on colistin 2 * 4.5 million IU. After the colistin infusion, biochemical tests were requested to control the patient's clinical situation. CK-MB mass and ProBNP values were measured in high concentrations. Cardiology consultation was requested to evaluate the increase in the CK-MB mass and ProBNP values. The patient's ECG and echocardiography showed no abnormality. The increase in cardiac markers was neither clinically acceptable nor insignificant. There was no hemolysis in the sample or analytical error in the device. Variability in the tests was thought to be due to the interference. As the bloodletting time was questioned, it was determined that it was taken during colistin treatment. In order to determine the effect of colistin-related interference on the other tests, the laboratory was contacted and additional tests (TSH, FT4, Anti- TPO, B-HCG, Estradiol, Prolactin, CA 125, CA 15-3, CA 19-9, Vitamin B12, C-Peptide, DDimer, PTH, 25 hydroxy vitamin D, PT, INR, APTT) were conducted. During colistin treatment, in many tests, bias was detected between -75 and + 268.80%. CONCLUSION: Clinicians should consider suspicious test results that are incompatible with the diagnosis for the possibility of erroneous measurements due to colistin interference and review the sampling processes.

Colistin Induced Assortment of Antimicrobial Resistance in a Clinical Isolate of Acinetobacter baumannii SD01.

BACKGROUND: Colistin was considered as the most effective antibiotic against Acinetobacter baumannii, a widely-known opportunistic pathogen. In recent years, a number of colistin resistant strains have also been reported. OBJECTIVE: This work is commenced to investigate the contribution of efflux pumps toward resistance to colistin-like cyclic polypeptide antibiotics, since the efflux pumps serve as the escape routes leading to drug-resistance. METHODS: RNA was extracted from A. baumannii isolates cultured from samples procured by tracheal aspiration of infected patients. The expressions of gene(s) that played major roles in the regulation of efflux pump families and involvement of integron systems were studied using real time PCR. Antimicrobial susceptibility tests were conducted to investigate antibiotic resistance of the isolates. RESULTS: It was observed that genes coding for sugE, ydhE, ydgE, mdfA, ynfA and tolC significantly contributed to resistance against colistin antibiotics, however, no significant transcriptional change was observed in the efflux pump, MexAB-OprM. Results suggest that A. baumanii readily pumps out colistin via efflux pumps belonging to MATE and SMR family. CONCLUSION: Integral role of efflux pumps and integron 1 genetic system was elucidated towards evolution of multi-drug resistant strain(s). Therefore, for accurate therapeutics, an early detection of efflux genes is crucial before prescribing against colistin resistant A. baumanii.

Elevated serum procalcitonin predicts Gram-negative bloodstream infections in patients with burns.

BACKGROUND: Many studies have suggested that procalcitonin can predict bloodstream infection and also distinguish between Gram-negative, Gram-positive and fungal infections after burn. However, up to now, there is no literature on serum procalcitonin level of multidrug-resistant pathogens and non-multidrug-resistant pathogens among Gram-negative bloodstream infections after burn. The purpose of this study is to explore the value of serum procalcitonin in identifying Gram-negative bloodstream infection in patients with febrile critical burn and then to investigate the difference of serum procalcitonin level between multidrug-resistant pathogens and non-multidrug-resistant pathogens among Gram-negative bloodstream infections after burn. METHODS: Patients with febrile critical burn admitted to the burn department of our hospital from 1 January 2014 to 1 August 2018 were retrospectively analysed. Patients with positive blood culture whose blood samples were collected for simultaneous blood culture and procalcitonin testing were enrolled. All strains were identified by an automatic microorganism analyser, and procalcitonin was analysed by an automatic electrochemiluminescence immunoassay. RESULTS: Overall, a total of 119 patients with positive blood culture met the inclusion criteria. There were 64 Gram-negative bacilli, 38 Gram-positive bacteria, 8 C. albicans and 9 polymicrobial bloodstream infections. The median procalcitonin value in Gram-negative bloodstream infections (2.67 ng/mL, interquartile range (IQR) 1.58-6.08) was significantly higher than that in Gram-positive bloodstream infections (1.04 ng/mL, IQR 0.35-1.60, P < 0.01), or C. albicans bloodstream infections (1.09 ng/mL, IQR 0.82-2.30, P < 0.05). Receiver operating characteristic curve (ROC) analysis showed that in addition to polymicrobial bloodstream infections, the area of procalcitonin under the curve distinguishing Gram-negative bloodstream infections from all other blood culture-positive bloodstream infections was 0.761, the best critical value was 1.73 ng/mL, the sensitivity was 73%, the specificity was 74%, the positive predictive value was 80%, the negative predictive value was 67%, The level of procalcitonin was significantly higher in multidrug-resistant Gram-negative bacilli (A. baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa) (2.76 ng/mL, IQR 2.01-7.76) than in non-multidrug-resistant bacilli (1.01 ng/mL, IQR 0.58-1.56, P < 0.01). CONCLUSION: Elevated serum procalcitonin can identify Gram-negative bloodstream infections in patients with febrile critical burn. In Gram-negative bloodstream infections, high procalcitonin levels may be associated with multidrug-resistant Gram-negative bacilli (A. baumannii, K. pneumoniae and P. aeruginosa).

Sepsis 2019: What Surgeons Need to Know.

The definition of sepsis continues to be as dynamic as the management strategies used to treat this. Sepsis-3 has replaced the earlier systemic inflammatory response syndrome (SIRS)-based diagnoses with the rapid Sequential Organ Failure Assessment (SOFA) score assisting in predicting overall prognosis with regards to mortality. Surgeons have an important role in ensuring adequate source control while recognizing the threat of carbapenem-resistance in gram-negative organisms. Rapid diagnostic tests are being used increasingly for the early identification of multi-drug-resistant organisms (MDROs), with a key emphasis on the multidisciplinary alert of results. Novel, higher generation antibiotic agents have been developed for resistance in ESKCAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) organisms while surgeons have an important role in the prevention of spread. The Study to Optimize Peritoneal Infection Therapy (STOP-IT) trial has challenged the previous paradigm of length of antibiotic treatment whereas biomarkers such as procalcitonin are playing a prominent role in individualizing therapy. Several novel therapies for refractory septic shock, while still investigational, are gaining prominence rapidly (such as vitamin C) whereas others await further clinical trials. Management strategies presented as care bundles continue to be updated by the Surviving Sepsis Campaign, yet still remain controversial in its global adoption. We have broadened our temporal and epidemiologic perspective of sepsis by understanding it both as an acute, time-sensitive, life-threatening illness to a chronic condition that increases the risk of mortality up to five years post-discharge. Artificial intelligence, machine learning, and bedside scoring systems can assist the clinician in predicting post-operative sepsis. The public health role of the surgeon is key. This includes collaboration and multi-disciplinary antibiotic stewardship at a hospital level. It also requires controlling pharmaceutical sales and the unregulated dispensing of antibiotic agents globally through policy initiatives to control emerging resistance through prevention.

Multiple sequence types responsible for healthcare-associated Acinetobacter baumannii dissemination in a single centre in Egypt.

BACKGROUND: Acinetobacter baumannii is an increasingly worrying organism in the healthcare setting, due to its multidrug resistance and persistence. Prolonged hospitalisation, immunocompromised patients and excessive antibiotic exposure all contribute to increasing the risk of A. baumannii infections, which makes cancer patients a significant risk group. This study aims to investigate the dissemination of A. baumannii at the National Cancer Institute (NCI) in Cairo - Egypt. METHODS: All bacterial isolates were typed using Multi-locus Sequence Typing (MLST) to characterise the epidemiology of isolates. The intrinsic OXA-51-like, and the acquired carbapanemases OXA-23, - 24/40, - 58, NDM, IMP, and VIM were also amplified and sequenced to genetically identify mechanisms of carbapenem resistance. RESULTS: MLST results show a high degree of multi-clonal dissemination, with 18 different Sequence Types (STs) identified, including 5 novel. The majority of isolates belonged to International Clone (IC) 2, and carbapenem resistance was detected in 93% of isolates and mediated by blaOXA-23, blaOXA-58, blaNDM-1 and blaVIM-1. We also report the presence of a resistant ST732 (OXA-378) which has been previously identified in migratory birds. CONCLUSIONS: Multiple highly resistant clones were identified in a Cancer hospital in Cairo. It is vital that clinicians and healthcare workers are aware of the population of A. baumannii present in order to have appropriate treatment and infection control practices.

An interactive nomogram to predict healthcare-associated infections in ICU patients: A multicenter study in GuiZhou Province, China.

OBJECTIVE: To develop and validate an interactive nomogram to predict healthcare-associated infections (HCAIs) in the intensive care unit (ICU). METHODS: A multicenter retrospective study was conducted to review 2017 data from six hospitals in Guizhou Province, China. A total of 1,782 ICU inpatients were divided into either a training set (n = 1,189) or a validation set (n = 593). The patients' demographic characteristics, basic clinical features from the previous admission, and their need for bacterial culture during the current admission were extracted from electronic medical records of the hospitals to predict HCAI. Univariate and multivariable analyses were used to identify independent risk factors of HCAI in the training set. The multivariable model's performance was evaluated in both the training set and the validation set, and an interactive nomogram was constructed according to multivariable regression model. Moreover, the interactive nomogram was used to predict the possibility of a patient developing an HCAI based on their prior admission data. Finally, the clinical usefulness of the interactive nomogram was estimated by decision analysis using the entire dataset. RESULTS: The nomogram model included factor development (local economic development levels), length of stay (LOS; days of hospital stay), fever (days of persistent fever), diabetes (history of diabetes), cancer (history of cancer) and culture (the need for bacterial culture). The model showed good calibration and discrimination in the training set [area under the curve (AUC), 0.871; 95% confidence interval (CI), 0.848-0.894] and in the validation set (AUC, 0.862; 95% CI, 0.829-0.895). The decision curve demonstrated the clinical usefulness of our interactive nomogram. CONCLUSIONS: The developed interactive nomogram is a simple and practical instrument for quantifying the individual risk of HCAI and promptly identifying high-risk patients.

Bloodstream infections caused by carbapenem-resistant Acinetobacter baumannii: Clinical features, therapy and outcome from a multicenter study.

OBJECTIVES: bloodstream infections (BSI) due to multidrug-resistant (MDR) Acinetobacter baumannii (AB) have been increasingly observed among hospitalized patients. METHODS: prospective, observational study conducted among 12 large tertiary-care hospitals, across 7 Italian regions. From June 2017 to June 2018 all consecutive hospitalized patients with bacteremia due to MDR-AB were included and analyzed in the study. RESULTS: During the study period 281 episodes of BSI due to MDR-AB were observed: 98 (34.8%) episodes were classified as primary bacteremias, and 183 (65.2%) as secondary bacteremias; 177 (62.9%) of them were associated with septic shock. Overall, 14-day mortality was observed in 172 (61.2%) patients, while 30-day mortality in 207 (73.6%) patients. On multivariate analysis, previous surgery, continuous renal replacement therapy, inadequate source control of infection, and pneumonia were independently associated with higher risk of septic shock. Instead, septic shock and Charlson Comorbidity Index >3 were associated with 14-day mortality, while adequate source control of infection and combination therapy with survival. Finally, septic shock, previous surgery, and aminoglycoside-containing regimen were associated with 30-day mortality, while colistin-containing regimen with survival. CONCLUSIONS: BSI caused by MDR-AB represents a difficult challenge for physicians, considering the high rates of septic shock and mortality associated with this infection.

Acinetobacter baumannii meningitis in children: a case series and literature review.

INTRODUCTION: The incidence of Acinetobacter baumannii meningitis, which typically occurs after neurosurgery, has increased in recent years. Pediatric Acinetobacter baumannii meningitis due to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has important clinical significance. METHODOLOGY: We retrospectively reviewed the clinical course and outcome of nine cases of meningitis due to Acinetobacter baumannii in children and reviewed the relevant literature. RESULTS: Seven patients had a history of neurosurgery, and the average time from the first surgery to cerebrospinal fluid (CSF) culture in these seven patients was 23.71 ± 17.43 days. Of all nine patients, four patients showed MDR isolates, two showed XDR isolates, and one showed pan-drug-resistant (PDR) isolates. Three patients received an intrathecal injection of amikacin. Two patients received intravenous colistin (5 mg/kg), and one received polymyxin B (2 mg/kg). The mean hospitalization duration was 39.44 days. Four patients eventually died: two with MDR Acinetobacter, one with PDR Acinetobacter, and one with susceptible Acinetobacter. Two of them still had positive CSF cultures at death. CONCLUSION: Acinetobacter baumannii meningitis is usually associated with neurosurgery and the placement of foreign material, and it usually has a high mortality. Intrathecal or intraventricular polymyxin administration is expected to be an effective choice for meningitis but requires further study.

Exploring a phage-based real-time PCR assay for diagnosing Acinetobacter baumannii bloodstream infections with high sensitivity.

In the present study, we developed a phage-based real-time quantitative PCR (qPCR) methodology for sensitive diagnosis of bloodstream infection (BSI) caused by Acinetobacter baumannii (A. baumannii). An isolated A. baumannii phage p53 was used for Taqman qPCR through detecting phage replication in live A. baumannii cells in serum samples. At the phage concentration of 103 PFU/mL, the sensitive detection of A. baumannii (down to 10 CFU in 100 µL serum) has been obtained within 4 h in spiked serum samples without bacteria isolation and DNA extraction. Subsequent testing of 22 simulated serum samples spiked by different strains has shown that the results from the phage-based Taqman qPCR method have 100% agreement with the spiked concentrations of the bacteria. The assay built in this study, gathering all the advantages for detections of high rapidity, high sensitivity, good specificity, being able to detect only live bacteria not dead bacteria and no DNA extraction or purifications, can be developed to detecting other bacterial pathogens in serum or other complicated samples through switching to other types of phages and realize the rapid and sensitive detection of bacteria in BSI, which would potentially be applied for fast diagnosis in sepsis clinically.

Isolation and identification of Acinetobacter spp. from healthy canine skin.

BACKGROUND: Acinetobacter species can exhibit widespread resistance to antimicrobial agents. They are already recognized as important nosocomial pathogens of humans, but are becoming increasingly recognized in opportunistic infections of animals. HYPOTHESIS/OBJECTIVES: This study aimed to determine whether Acinetobacter spp. are carried on skin of healthy dogs and, if present, to identify the species. ANIMALS: Forty dogs were sampled at veterinary practices and rescue centres. They were free from skin disease and receiving no systemic or topical treatments. METHODS: Skin swab samples were collected from four sites on each dog and cultured. Acinetobacter spp. isolates were detected by biochemical tests and gas chromatography. The species was determined by sequencing the RNA polymerase ß-subunit (rpoB) gene. Isolates were screened for OXA carbapenemase genes and class 1 integrons capable of carrying resistance genes, and subjected to antimicrobial susceptibility tests. RESULTS: For 25% dogs sampled (10 of 40), Acinetobacter spp. were isolated at one or more skin sites. Thirteen Acinetobacter spp. isolates were recovered from 160 samples. The most frequently cultured was A. lwoffii (seven of 13), followed by A. baumannii (two of 13), A. junii (one of 13), A. calcoaceticus (one of 13), A. pittii (one of 13) and a novel Acinetobacter species (one of 13). Class 1 integrons and blaOXA-23-like were not detected. Isolates were susceptible to most antibiotics. CONCLUSIONS AND CLINICAL IMPORTANCE: The study confirms that Acinetobacter spp. can survive on canine skin, where they may be potential reservoirs for infection. This highlights the importance of good hygiene in veterinary practice, adhering to aseptic principles in surgery, and treatment based on culture and susceptibility testing where possible.

Microbiological Characteristics and Clinical Outcomes of Acinetobacter spp. Endophthalmitis with the First Reported Case of Acinetobacter haemolyticus Endophthalmitis.

AIM: To present the microbiological details and clinical outcomes of Acinetobacter spp. endophthalmitis with the first reported case of A. haemolyticus. METHODS: A retrospective study of microbiologically proven Acinetobacter spp. endophthalmitis was carried out from 2010 to 2015. The data collected included age, type of endophthalmitis, best-corrected visual acuity (BCVA), species involved, and antibiotic susceptibility pattern. The primary outcomes measured were anatomical outcomes in terms of globe integrity and functional outcome as BCVA at last follow-up. RESULTS: Eleven patients out of 3004 patients who underwent surgery for endophthalmitis were due to Acinetobacter spp. Seven cases (63.6%) were both smear and culture positive; there were two cases (18%) each of A. haemolyticus and A. baumanii. Four cases (36%) were only culture positive with negative smear. Three cases (27.2%) were polymicrobial. Ten cases (91%) were susceptible to amikacin and nine (82%) to ciprofloxacin. Six (54.5%) were resistant to ceftazidime. Mean logMAR BCVA improved to 1.8 (20/1330) from an initial 2.5 (20/6839). Pthisis bulbi was seen in two cases (18%). CONCLUSIONS: Even though the outcomes of Acinetobacter spp. endophthalmitis are modest to poor, outcomes following intervention are relatively good for A. haemolyticus. These cases have good susceptibility to amikacin, but are often resistant to ceftazidime.

Infected Nonhealing Wound in a Kidney Transplant Recipient: Successful Treatment With Topical Homologous Platelet-Rich Gel.

Impaired would healing is a known adverse effect of chronic immunosuppression. Solid-organ transplant recipients undergoing major abdominal surgery have an increased risk of wound-related complications compared with the general population. In this subset of patients, surgical site infections and wound dehiscence must be aggressively treated to avoid sepsis, graft loss, and death. Recently, topical application of platelet-rich plasma has been proposed as an alternative therapeutic option to enhance wound healing in difficult cases. Unfortunately, randomized controlled trials evaluating the efficacy of platelet-rich plasma compared with standard or advanced wound management are lacking, and the literature mostly refers to anecdotal reports in patients with no evidence of wound infection. This report documents a kidney transplant recipient who experienced spontaneous bladder rupture because of gangrenous cystitis. After an exploratory laparotomy and bladder repair, the patient developed a deep surgical site infection by multidrug resistant Acinetobacter baumannii and extensive wound dehiscence. Advanced wound management and vacuum-assisted closure therapy were ineffective. Topical homologous platelet-rich gel was used resulting in significant wound healing, without infections or immunologic complications.

Bronchoscopic Drainage of Lung Abscesses Using a Pigtail Catheter.

BACKGROUND: Lung abscesses are commonly treated with antibiotics. However, some patients fail to respond and may require percutaneous catheter drainage or surgical intervention. Bronchoscopic drainage (BD) of lung abscesses emerged as a therapeutic alternative in selected patients. OBJECTIVE: To describe our experience with 15 patients who underwent BD at our center during 2006-2016. METHODS: Patients underwent flexible bronchoscopy. Under fluoroscopic guidance, a pigtail catheter was introduced into the abscess cavity, its correct position being confirmed by the injection of contrast medium. The catheter remained in place for a few days and was flushed repeatedly with antibiotics. RESULTS: Fifteen patients (9 males; median age 59 years) underwent 16 BD procedures. A pigtail catheter was successfully inserted and pus was drained from the abscess cavity in 13 procedures (81%) conducted in 12 patients, leading to rapid clinical improvement in 10 of them; resolution of fever occurred a median of 2 days (range <1-4) following pigtail insertion, and patients were discharged after 8 days (range 4-21). The pigtail catheter was extracted after a median of 4 days (range 2-6). CONCLUSION: BD of lung abscesses was achieved in 13 out of 16 procedures, leading to rapid improvement in the majority of patients. This work adds to the existing literature in establishing this procedure as an acceptable therapeutic alternative in selected patients who fail to respond to antibiotics, especially those with an airway obstruction or a fairly central lung abscess.

Bacteremia due to Acinetobacter ursingii in infants: Reports of two cases.

Acinetobacter ursingii is an aerobic, gram-negative, opportunistic microorganism which is rarely isolated among Acinetobacter species. We present two immunocompetent infants who developed bacteremia due to A. ursingii. The first patient is a two -month- old boy who had been hospitalized in pediatric surgery unit for suspected tracheo-esophageal fistula because of recurrent aspiration pneumonia unresponsive to antibiotic therapy. The second patient is a fourteen -month- old boy with prolonged vomiting and diarrhea. A. ursingii was isolated from their blood cultures. They were successfully treated with ampicillin-sulbactam. Although A. ursingii has recently been isolated from a clinical specimen; reports of infection with A. ursingii in children are rare. A. ursingii should be kept in mind as an opportunistic microorganism in children.

Retrospective Study on Multidrug-Resistant Bacterium Infections After Rigid Internal Fixation of Mandibular Fracture.

PURPOSE: To retrospectively investigate infection by multidrug-resistant bacteria (MDRB) after rigid internal fixation (RIF) of mandibular fracture and determine risk factors and cure methods. PATIENTS AND METHODS: From 2009 through 2014, 933 patients with mandibular fracture were enrolled in the study. Fifteen variables were statistically analyzed using univariate and multivariate logistic regression methods to investigate risk factors for MDRB infection after RIF of mandibular fracture. RESULTS: Sixteen of 933 patients (1.71%) developed MDRB infection. Of these, 6 were infected with methicillin-resistant Staphylococcus aureus (MRSA), 6 with multidrug-resistant Pseudomonas aeruginosa (MDR-PA), 1 with extended spectrum ß-lactamase-producing Klebsiella pneumonia, 1 with extended spectrum ß-lactamase-producing Escherichia coli, 1 with multidrug-resistant Acinetobacter baumannii, and 1 with multidrug-resistant Enterobacter cloacae. Univariate analysis showed that risk factors of MDRB infection after RIF of mandibular fracture were age, obesity (body mass index ≥25 kg/m(2) for Asians), polytrauma (Injury Severity Score >16), preoperative infection, open fractures, comminuted fractures accompanied by other facial fractures, and teeth involving the fracture line. Multivariate logistic regression analysis showed that obesity, preoperative infection, and open fractures were independent risk factors of MDRB infection. After systemic anti-infection treatments with vancomycin, piperacillin, tazobactam, local drainage, and debridement, the infections were under control. Fourteen patients achieved clinical healing at an average time of 8.71 months, and 2 did not achieve clinical healing. The overall mean follow-up was 18.81 months. CONCLUSION: MDRB infections occurring after RIF of mandibular fracture were caused mainly by MRSA and MDR-PA. Obesity, preoperative infection, and open fractures were the main risk factors. To lower risk, surgical debridement should be performed sooner once acute infection has been controlled, stable fixation should be achieved with the smallest amount of internal fixation material, the blood supply should be protected, and a reconstruction plate should be used to fix sites with greater stress or large comminuted fracture.