Multiplex PCR for Detection of a Successful Pathogen; Acinetobacter baumanni as a Real Threat in Intensive Care Unit of a University Hospital

Multiplex PCR for Detection of a Successful Pathogen; Acinetobacter baumanni as a Real Threat in Intensive Care Unit of a University Hospital

To analyze the detection, drug resistance, and status of infection of Acinetobacter baumannii (AB) in burn ICU during 3 years. A total of 2 010 specimens of wound secretion, blood, venous catheter attachment, sputum, stool and urine were collected from 505 burn patients hospitalized in our burn ICU from January 2011 to December 2013, and bacterial culture was performed. Pathogens were identified by automatic microorganism identifying and drug sensitivity analyzer. Drug resistance of all the obtained AB to 16 antibiotics commonly used in clinic, including cefoperazone/sulbactam, polymyxin, etc., was tested with K-B paper disk diffusion method. Patients with AB infection were ascertained. The WHONET 5.6 software was used to analyze the distribution of pathogens during 3 years, the isolation of AB with different sources and the status of drug resistance of AB to 16 antibiotics each year, and the status of patients with AB infection, and their outcome. A total of 961 strains of pathogens were isolated, among which 185 (19.25%) strains were Gram positive cocci, 728 (75.75%) strains were Gram negative bacilli, and 48 (4.99%) strains were fungi. A total of 172 strains of AB were isolated, ranking the second place among all the detected pathogens, with 67 (38.95%) strains from wound secretion, 11 (6.40%) strains from blood, 23 (13.37%) strains from venous catheter attachment, and 71 (41.28%) strains from sputum, no AB strain was isolated from feces or urine. The AB strains were found sensitive to polymyxin and with relatively low drug resistance rate to minocycline, while the drug resistance rates were over 80.0% to the other 14 antibiotics commonly used in clinic in 2013. AB culture of wound secretion was positive in 27 patients. Among them, 7 patients suffered from wound infection, and the wound infection was caused by AB in 1 out of the 7 patients. AB culture of blood was positive in 7 patients. Among them, 3 patients suffered from bloodstream infection, and the infection was due to AB invasion in 1 out of the 3 patients. AB culture of venous catheter attachment was positive in 20 patients. Among them, 8 patients suffered from bloodstream infection, and the infection was due to AB invasion in 1 out of the 8 patients. AB culture of sputum was positive in 35 patients. Among them, 13 patients suffered from ventilatory associated pneumonia, and 2 out of the 13 patients were diagnosed as AB infection. A total of 69 patients were AB culture positive, among them 64 patients were cured, 2 patients were transferred to other hospitals, and 3 patients died, with the mortality rate of 4.35%. AB in our burn ICU has a high detection rate and extensive drug resistance in above-mentioned 3 years. However, AB was mainly colonized in patients with extensive burns with a low mortality rate.

To explore the clinical utility of multiple polymerase chain reaction (M-PCR) in the rapid detection of the common pathogens in ventilator-associated trachea - bronchitis (VAT) and ventilator-associated pneumonia (VAP). Sputum samples of 75 patients complicated VAT or VAP in surgical intensive care unit (SICU), were examined by bacterial culture, ordinary PCR, the M-PCR detection. The pathogen detection rates among three methods were compared. The Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae of the positive detection rates were 50.7%, 45.3%, 30.7%, 41.3% and 58.7% by bacterial culture. By ordinary PCR, the positive detection rates were respectively 88.0%, 89.3%, 78.7%, 85.3% and 93.3%, and by M-PCR, the positive detection rates were respectively 92.1%, 90.7%, 82.7%, 89.3% and 96.0%. The positive rates of five common pathogens of ordinary PCR and M-PCR were higher than those of bacterial culture (all P < 0.05). The M-PCR had merit for rapid detection compared with ordinary PCR. Compared with bacterial culture, ordinary PCR and M-PCR yield higher positive rates in identifying five common pathogens of VAT and VAP, meanwhile, it also demonstrated the tendency that M-PCR may save cost and labor power.

Objective To investigate the incidence of Acinetobacter baumannii induced paediatric bacterial meningitis and to explore the characteristics, treatment, and prognosis of Acinetobacter baumannii meningitis. Methods The cerebrospinal fluid specimens and the clinical data about patients who acquired intracranial infection of Acinetobacter baumannii were collected from 10 children′s hospital, and analysed by WHONET. Results A total of 318 positive cerebrospinal fluid specimens from 10 hospitals were collected, and 16(5%) of Acinetobacter baumannii were detected.Acinetobacter baumannii was completely resistant to Aztreonam(100%), Cefotetan(100%) and Cefazolin(100%), and the resistance rates of Acinetobacter baumannii to Ceftriaxone and Ciprofloxacin were above 80%, and the resistance rates of Acinetobacter baumannii to Ceftazidime and Cefepime were all 68.8%, and the resistance rates of Acinetobacter baumannii to Meropenem, Imipenem, Cefoperazone/Sulbactam were 69.2%, 68.8% and 53.3%, respectively.In 16 cases of intracranial infection caused by Acinetobacter baumannii, 11 cases(68.8%) had underlying diseases such as intracranial tumor, hydrocephalus, craniocerebral injury and intracranial hemorrhage, and all 11 cases underwent surgical intervention, such as surgical resection and hydrocephalus drainage.Among 16 cases, 14 cases(87.5%) were admitted to SICU, PICU or NICU and other intensive care units, and the lengths of stay were all >20 d. Under the combined treatment of multiple antibiotics, 16 cases of intracranial infection caused by Acinetobacter baumannii, 6 cases died, with a mortality of 37.5%. Conclusion Acinetobacter baumannii meningitis is becoming an increasingly common clinical entity recently.Because of the resistance to antibiotics, the therapy for Acinetobacter baumannii infection encounters arduous challenge.Insisting on multi-center monitoring and using antibiotics reasonably and effectively appears to be particularly important. Key words: Acinetobacter baumannii; Bacterial meningitis; Cerebrospinal fluid; Children; Clinical analysis

ObjectiveTo evaluate the diagnostic performance of metagenomic next-generation sequencing (mNGS) and culture in pathogen detection among intensive care unit (ICU) and non-ICU patients with suspected pulmonary infection.MethodsIn this prospective study, sputum samples were collected from patients with suspected pulmonary infection for 2 consecutive days and then subjected to DNA or RNA sequencing by mNGS or culture; 62 ICU patients and 60 non-ICU patients were admitted. In the end, comparisons were made on the pathogen species identified by mNGS and culture, the overall performance of these two methods in pathogen detection, and the most common pathogens detected by mNGS between the ICU and non-ICU groups.ResultsIn DNA and RNA sequencing, the positive rate of pathogen detection reached 96.69% (117/121) and 96.43% (108/112), respectively. In culture tests, the positive rate of the pathogen was 39.34% (48/122), much lower than that of DNA and RNA sequencing. In general, the positive rate of pathogen detection by sputum mNGS was significantly higher than that of sputum culture in the total and non-ICU groups (p < 0.001) but did not show a significant difference when compared to the result of sputum culture in the ICU group (p = 0.08). Haemophilus spp., Candida albicans, Enterococcus spp., and viruses from the mNGS results were excluded before comparing the overall performance of these two methods in pathogen detection. Specifically, among the 10 most common bacteria implied from the mNGS results, significant differences were observed in the number of cases of Haemophilus parainfluenzae, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Staphylococcus aureus, and Enterococcus faecalis between the ICU and non-ICU groups (p < 0.05).ConclusionsThis study demonstrated the superiority of mNGS over culture in detecting all kinds of pathogen species in sputum samples. These results indicate that mNGS may serve as a valuable tool to identify pathogens, especially for ICU patients who are more susceptible to mixed infections.

Objective To study the infection situation of acinetobacter baumannii and the drug susceptibility in intensive care unit (ICU), and to provide reference for rational use of antibiotics in clinic. Methods A retrospective study was conducted.acinetobacter baumannii separated in ICU of Erduosi Central Hospital from January 2013 to December 2015 were collected.In the study of these 3 years in ICU, the situation of acinetobacter baumannii infection and the acinetobacter baumannii susceptibility to drugs were analyzed. Results One hundred and two strains acinetobacter baumannii were isolated in 3 years, acinetobacter baumannii was mainly isolated in sputum samples in 77 cases which accounted for 75.5%.And other 10 strains were isolated in urine, accounted for 9.8%.7 strains were isolated in secretion, which accounted for 6.9%.The isolation rate of acinetobacter baumannii was increased year by year.The highest rate of susceptibility was to tigecycline, with a susceptibility rate of 95.1%, followed by cefoperazone-sulbactm, with the rate of 40.2%, and to imipenem was 35.3%.Susceptibility of acinetobacter baumannii isolated was less than 40.0% for antibiotic commonly used. Conclusion In ICU, the rate of acinetobacter baumannii infection increased annually and the infection was mainly in the respiratory tract.The susceptibility of tigecycline to acinetobacter baumannii was the highest, and its susceptibility to clinical commonly used antibiotics was poor. Key words: Infection; Intensive care unit; Microbial sensitivity tests

Objective To investigate antibiotics resistant characteristics and carbapenemases genotype of Acinetobacter baumannii in Intensive Care Unit (ICU), so as to provide theoretical basis for clinical prevention and treatment. Methods Retrospective study was made on 90 non-duplicated clinical isolates of Acinetobacter baumannii, which were collected From January 2013 to January 2014 in three tertiary hospitals of Qingdao. All strains were identified by VITEK2 automated microbiology analyzer; K-B method was used to do drug susceptibility test; polymerase chain reaction (PCR) was used to amplify the OXA-23, OXA-24, OXA-51, OXA-58, KPC-2, VIM, IMP genes, and the positive products of genes were sequenced; the chi-square test was used to compare the difference of the resistance rates. Results The detection rate of multi-drug resistant A. baumannii (MDRAB)and Pan-drug resistant A. baumannii (PDRAB)was 61.11% (55/90) and 17.78% (16/90). In the 32 strains of imipenem-resistant Acinetobacter baumannii, the resistant rates to Cefoperazone/sulbactam, Polymyxin B was lower, while the resistant rates to other drugs tested were more than 85%. The difference of the resistance rates to 9 drugs between imipenem resistant group and Imipenem sensitive group were statistically significant (P≤0.05). PCR result showed: 32 strains detected OXA-51 gene, 28 strains detected OXA-23 gene, and 3 strains detected VIM gene, the detection rates of which were 100%, 87.50% and 9.38% respectively.All strains were not detected OXA-24, OXA-58, KPC-2 and IMP genes. The sequenced results were absolutely homology with the corresponding genes in genbank. Conclusions The resistance of A. baumannii in ICU is serious in this region, especially imipenem-resistant A. baumannii, which were nearly no-sensitive to most of the drugs commonly used in clinical. The gene existence of carbapenemase and carbapenemase producing is one of the main resistance mechanism of Acinetobacter baumannii to carbapenem antibiotics. OXA-23 was the major genotypes in this region.(Chin J Lab Med, 2017, 40: 216-220) Key words: Acinetobacter baumannii; Drug resistance, bacterial; Bacterial proteins; Beta-Lactamases; Genotype; Intensive care units

The present study aimed to investigate the association between drug resistance and class I, II and III integrons in Acinetobacter baumannii (ABA). Multilocus sequence typing (MLST) is a tool used to analyze the homology among house-keeping gene clusters in ABA and ABA prevalence and further provides a theoretical basis for hospitals to control ABA infections. A total of 96 clinical isolates of non-repeating ABA were harvested, including 74 carbapenem-resistant ABA (CRABA) and 22 non-CRABA strains, and used for bacterial identification and drug susceptibility analysis. Variable regions were sequenced and analyzed. Then, 7 pairs of housekeeping genes were amplified and sequenced via MLST and sequence alignment was performed against the Pub MLST database to determine sequence types (STs) strains and construct different genotypic evolutionary diagrams. The detection rate of CRABA class I integrons was 13.51% (10/74); no class II and III integrons were detected. However, class I, II and III integrons were not detected in non-CRABA strains. The variable regions of 9 of 10 class I integrons were amplified and 10 gene cassettes including aacC1, aac1, aadDA1, aadA1a, aacA4, dfrA17, aadA5, aadA1, aadA22 and aadA23 were associated with drug resistance. The 96 ABA strains were divided into 21 STs: 74 CRABA strains containing 9 STs, primarily ST208 and ST1145 and 22 non-CRABA strains containing 18 STs, primarily ST1145. Class I integrons are a critical factor underlying drug resistance in ABA. CRABA and non-CRABA strains differ significantly; the former primarily contained ST208 and ST1145, and the latter contained ST1145. Most STs were concentrated in intensive care units (ICUs) and the department of Neurology, with the patients from the ICUs being the most susceptible to bacterial infection. In the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital, ABA is potentially horizontally transmitted and MLST can be used for clinical ABA genotyping.

Objective To investigate the infection and drug resistance of Acinetobacter baumannii in the Third Affiliated Hospital of Sun Yat-sen University. Methods Clinical data of 209 patients infected with Acinetobacter baumannii in the Third Affiliated Hospital of Sun Yat-sen University between June 2012 and December 2014 were retrospectively analyzed. Among the patients, 160 were males and 49 were females with the average age of (55±18) years old. The informed consents of all patients were obtained and the local ethical committee approval was received. The specimen source, ward distribution and drug resistance of Acinetobacter baumannii were observed. The comparison of drug resistance was conducted using Chi-square test and Bonferroni test. Results Two hundred and thirty-six strains of Acinetobacter baumannii were isolated in the hospital, and most were isolated from Department of Liver Transplantation (50 strains, 21.2%), the next were Department of Rehabilitation (34 strains, 14.4%) and Surgical Intensive Care Unit (SICU) (21 strains, 8.9%). Sputum specimens were the main source of Acinetobacter baumannii, respectively accouting for 84%, 76% and 71% in the three wards, where Acinetobacter baumannii had high drug resistance to the common antibiotics used in clinical. The drug resistance rate to sulfamethoxazole of Acinetobacter baumannii in Department of Liver Transplantation was 76%, significantly higher than 33% of SICU (χ2=11.60, P<0.017). Conclusions Acinetobacter baumannii is found most in Department of Liver Transplantation, which is followed by Department of Rehabilitation and SICU. Sputum specimen is the main source of Acinetobacter baumannii. Acinetobacter baumannii detected in the three wards has a high drug resistance to common antibiotics. Key words: Acinetobacter baumannii; Infection; Multidrug resistance-associated proteins; Liver transplantation

Phenotypic and molecular characterization of Acinetobacter baumannii isolates causing lower respiratory infections among ICU patients

Objective To research the association between the prognosis of Acinetobacter baumannii infection and drug resistance among patients in the intensive care unit( ICU). Methods Eighty-four patients with Acine tobacter baumannii infection from February 2003 to April 2009 in Peking University Shenzhen Hospital ICU were retrospectively analyzed and divided into alive group(59 patients) and dead group(25 patients) by using 30-day mortality. The impact of drug resistant Acinetobacter baumannii on mortality was assessed by binary logistic regression. Results The mortality of patients with Acinetobacter baumannii infection was related to age, haematological malignancy, the Chanson Comorbidity Index, APACHEII score, multidrug-resistant Acinetobacter baumannii ( MDRAB) , carbapenem-resistant Acinetobacter baumannii (CRAB ) , septic shock and antimicrobial inappropriate therapy by univariate analysis( P < 0.05). Binary logistic regression revealed that CRAB( OR = 3. 360,95%CI: 1. 563 ～7.223,P< 0.05)and the Charison Comorbidity Index( OR = 1. 633,95% CI: 1. 201 ～2. 221 ,P <0.05) were independent risk factors. Conclusions The prognosis of Acinetobacter baumannii infection was closely correlated with drug resistance. The patients with CRAB infection had a higher mortality. Key words: Acinetobacter baumannii; Prognosis; Drug resistance; Drug resistance,multiple

Objective To investigate the changing characteristics and associated factors of pathogens which caused ventilator-associated pneumonia (VAP) in the respiratory care unit.Methods A retrospective analyses was used to investigate 79 cases of VAP in the respiratory care unit from 2005 to September 2011.The data were analyzed using SPSS 17.0 software,and x2 -test was used for the analysis of variance comparison of the distribution and changing of VAP pathogens among different years,different age groups and patients with different underlying diseases.Rank correlation analysis was used to describe the correlation between pathogen change and age.Results Among the seven years,the top five for the detection of pathogens were Pseudomonas aeruginosa (23.2％),Acinetobacter baumannii (15.9％),methicillin-resistant Staphylococcus aureus ( 13.2 ％ ),Escherichia bacilli ( 11.3％),Stenotrophomonas narrow food Aeromonas (8.6％).Differences in the distribution of pathogens among different years,different age groups,and different treatment groups were statistically significant.Conclusions In the recent seven years,the incidence of VAP caused by Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus in the respiratory care unit increases significantly,the distribution of pathogens may be associated with hospitalization time,age of the patients,and the use of antibiotics. Key words: Respiratory care unit; Ventilator-associated pneumonia; Pathogens

Background: Acinetobacter baumannii is a coccobacillus that is Gram negative, non motile, non fermentative and oxidase negative. It is the most common and successful nosocomial pathogen recognised by WHO. This dreadful pathogen causes urinary tract infections, ventilator associated pneumonia (VAP), bacteremia, etc., These infections are most common in hospital wards especially Intensive Care Unit (ICU). The infections are due to biofilm formation by the virulent genes of A. baumannii, and the common biofilm-associated genes of A. baumannii were bap, csuE, fimH, epsA, bfmS, ptk, pgaB, ompA, blaPER-1. Among these, bap, epsA and ompA genes are highly prevalent among the clinical strains of A. baumannii.&#x0D; Aim: To detect the three vital biofilm-associated genes of A. baumannii by in-silico PCR analysis.&#x0D; Materials and Methods: 19 isolates of A. baumannii were selected and 3 target genes, namely epsA, ompA and bap gene were used for the amplification process through in-silico PCR simulation tools. Evolutionary analysis was done for the ompA gene.&#x0D; Results: The epsA gene was expressed in 10.52% of the total strains selected with the highest occurrence of ompA gene as 57.89%. bap gene was not observed from the study strains included. From evolutionary analysis based on ompA distributed strains, the Acinetobacter baumannii SDF and Acinetobacter baumannii BJAB0715 might be the parental strains where the evolution of strains would have started. Through successive generations, the Acinetobacter baumannii MDR-ZJ06 and Acinetobacter baumannii TYTH-1 had become the multidrug resistant strains present in the environment.&#x0D; Conclusion: The findings of the study confirms the distribution of epsA and ompA genes among the 19 different strains of A. baumannii. The study suggests periodical monitoring of biofilm based virulence genes among the clinical strains and to curtail the A. baumannii infections.

Acinetobacter baumannii in ICU patients: A prospective study highlighting their incidence, antibiotic sensitivity pattern and impact on ICU stay and mortality

Objective In order to prevent the infection of Acinetobacter baumannii and use antibiotics rationally, the clinical infection and drug resistant data of multi-drug resistance Acinetobacter baumannii (MRAB) detected in intensive care unit (ICU) of Beijing Friendship Hospital from 2011 to 2013were analyzed. Methods This study is a retrospective study. One hundred and eighty five strains of MRAB were collected from the patients in ICU from January 2011 to December 2013. Identificationand antibiotic susceptibility of strains were determined with Vitek-2 Compact automatic bacteria identification system. The annual infection rate of MRAB was counted. PCR was used to detect the resistance genes. The clinical features of the patients with MRAB were analyzed. The average age, acute physiology and chronic health evaluation (APACHE) Ⅱ score, duration in ICU and mortality ratio of the MRAB patients were compared with the patients without MRAB. Rank-sum test was used to analyze the average age, APACHE Ⅱscore and duration in ICU. Chi-squared test was used to analyze the mortality ratio and annual infection rate. Results The average age [(67±17) vs (59±19) years old, Z=-5.365, P=0], APACHEⅡ score [(25.68±7.93) vs (17.62±8.39), Z=-14.821, P=0], duration in ICU [(27±29) vs (5±8) d, Z=-4.342, P=0] and mortality ratio [10.82% (53/185) vs 28.65% (147/1 359), χ2=45.92, P=0] of the patients infected by MRAB were significantly higher than those without the infection. The MRAB was found mostly in sputum and bronchial precipitates (83.78%, 155/185). Though detection rate reduced yearly and there was a significant reduction in 2013 compared with 2011 [11.07% (69/469) vs 8.37% (52/621), χ2=8.755, P=0.003], the drug resistant rate was in high level and did not show any change in the 3 years. OXA-23 and OXA-51 were detected in all MRAB. Conclusions The main drug resistant mechanism of MRAB in ICU is related to OXA-23. More active methods of control and prevention of MRAB should be used in elderly and severely pneumonic patients. Intensive disinfection and isolation measures can decrease MRAB detection rate. Combined antibiotics should be used in patients with MRAB infection. (Chin J Lab Med, 2015, 38: 55-58) Key words: Acinetobacter baumannii; Drug resistance, multiple, bacterial; Intensive care units