Reliability of a Screening Method Using Antibiotic Disks to Detect Carbapenemases in Glucose-Nonfermenting Gram-Negative Microorganisms From Clinical Samples of a Regional Hospital in Southeastern Spain.

BACKGROUND: Infections by glucose-nonfermenting gram-negative bacilli (NFGNB) pose a major public health problem due to multiresistance to beta-lactam antibiotics, especially plasmid-borne carbapenemases. Their detection by microbiology laboratories is challenging, and there is a need for easy-to-use and reliable diagnostic techniques. Our objective was to evaluate an in-house screening method to presumptively detect carbapenemases in NFGNB in a simple and clinically useful manner. METHODS: The study included 175 NFGNB isolates from urinary, respiratory, and rectal samples. In a triple assay, isolates were incubated at 37°C for 24 h on three solid-culture media: MacConkey II Agar, 5% Sheep Blood Columbia Agar and Mueller Hinton II Agar; meropenem (MEM) and cefepime (FEP) disks were employed for screening. Studies were then performed on the inhibition halo diameter, scanning effects, and the appearance of mutant colonies, which were compared with those observed using the colorimetric Neo-Rapid CARB Kit and immunochromatography (NG5-Test Carba and K-Set for OXA-23). Receiver operating characteristic curves were constructed for these data. RESULTS: Carbapenemases were expressed by 79/175 (45.1%): 19 Pseudomonas aeruginosa and 60 Acinetobacter baumannii. Optimal inhibition halo diameter cutoffs to detect this resistance on 5% sheep blood agar were as follows: 6 mm (MEM) and 6.5 mm (FEP) for P. aeruginosa (in the absence of scanning effects and mutations) and 10.5 mm (MEM) and 16 mm (FEP) for A. baumannii (even in the presence of scanning effects). CONCLUSION: The combined utilization of MEM and FEP antibiotic disks in 5% sheep blood agar, measuring their inhibition haloes, offers an effective method to predict the presence of carbapenemases as resistance mechanism in P. aeruginosa and A. baumannii.

Nomogram Analysis of Clinical Characteristics and Mortality Risk Factor of Non-Fermentative Gram-Negative Bacteria-Induced Post-Neurosurgical Meningitis.

Objective: To explore the clinical characteristics of post-neurosurgical meningitis (PNM) patients infected with nonfermenting Gram-negative bacilli (NFGNB) and to evaluate the related mortality risk factors. Methods: A cohort analysis of PNM patients infected with NFGNB in Beijing Tiantan Hospital and Capital Medical University from 2012.1 to 2020.12. The microbial distribution, antimicrobial sensitivity and genotypes were tested, and potential mortality risk factors were evaluated using Mann-Whitney U or chi-squared tests. Independent risk factors for mortality were established by constructing a logistic model. Results: A total of 2940 PNM patients were enrolled in this study, of whom 207 (17.1%) were infected with NFGNB. Among these patients, 29 died of NFGNB meningitis, with an overall mortality rate of 14.0%. The top three NFGNBs were Acinetobacter baumannii (105 cases, 50.7%), Pseudomonas aeruginosa (29 cases, 14.0%) and Acinetobacter lwoffii (20 cases, 9.7%). Nomogram analysis revealed that hypertension (OR 4.551, 95% CI: 1.464-14.154, P = 0.009), external ventricular drainage (EVD) (OR 3.944, 95% CI: 1.286-12.095, P = 0.016), and assisted mechanical ventilator (AMV) (OR 6.192, 95% CI: 1.737-22.081, P = 0.005) were independent risk factors for mortality. In addition, antibiotic prophylaxis was shown to play a vital role in NFGNB-induced PNM therapy. Conclusion: PNM patients infected with NFGNB have a high mortality rate. Hypertension, EVD and AMV were independent mortality risk factors, and clinical attention should be paid to their prevention and treatment.

Temporo-spatial variations in resistance determinants and clonality of Acinetobacter baumannii and Pseudomonas aeruginosa strains from Romanian hospitals and wastewaters.

BACKGROUND: Romania is one of the European countries reporting very high antimicrobial resistance (AMR) rates and consumption of antimicrobials. We aimed to characterize the AMR profiles and clonality of 304 multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa) strains isolated during two consecutive years (2018 and 2019) from hospital settings, hospital collecting sewage tanks and the receiving wastewater treatment plants (WWTPs) located in the main geographical regions of Romania. METHODS: The strains were isolated on chromogenic media and identified by MALDI-TOF-MS. Antibiotic susceptibility testing and confirmation of ESBL- and CP- producing phenotypes and genotypes were performed. The genetic characterization also included horizontal gene transfer experiments, whole-genome sequencing (WGS), assembling, annotation and characterization. RESULTS: Both clinical and aquatic isolates exhibited high MDR rates, especially the Ab strains isolated from nosocomial infections and hospital effluents. The phenotypic resistance profiles and MDR rates have largely varied by sampling point and geographic location. The highest MDR rates in the aquatic isolates were recorded in Galați WWTP, followed by Bucharest. The Ab strains harbored mostly blaOXA-23, blaOXA-24, blaSHV, blaTEM and blaGES, while Pa strains blaIMP, blaVIM, blaNDM, blaVEB, blaGES and blaTEM, with high variations depending on the geographical zone and the sampling point. The WGS analysis revealed the presence of antibiotic resistance genes (ARGs) to other antibiotic classes, such as aminoglycosides, tetracyclines, sulphonamides, fosfomycin, phenicols, trimethoprim-sulfamethoxazole as well as class 1 integrons. The molecular analyses highlighted: (i) The presence of epidemic clones such as ST2 for Ab and ST233 and ST357 for Pa; (ii) The relatedness between clinical and hospital wastewater strains and (iii) The possible dissemination of clinical Ab belonging to ST2 (also proved in the conjugation assays for blaOXA-23 or blaOXA-72 genes), ST79 and ST492 and of Pa strains belonging to ST357, ST640 and ST621 in the wastewaters. CONCLUSION: Our study reveals the presence of CP-producing Ab and Pa in all sampling points and the clonal dissemination of clinical Ab ST2 strains in the wastewaters. The prevalent clones were correlated with the presence of class 1 integrons, suggesting that these isolates could be a significant reservoir of ARGs, being able to persist in the environment.

Unravelling complex transposable elements surrounding blaGES-16 in a Pseudomonas aeruginosa ExoU strain.

OBJECTIVES: We characterised the complex surrounding regions of blaGES-16 in a Pseudomonas aeruginosa exoU+ strain (P-10.226) in Brazil. METHODS: Species identification was performed by MALDI-TOF MS, and the antimicrobial susceptibility profile was determined by broth microdilution based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The whole genome sequencing (WGS) of P-10.226 strain was performed using both short-read paired-end sequencing on the Illumina MiSeq platform as well as the long-read Oxford Nanopore MinION. RESULTS: WGS analysis showed that P-10.226 carried blaGES-16, which was found as a gene cassette inserted into a novel class I integron, In1992 (aadB-blaOXA-56-blaGES-16-aadB-aadA6c), whose 3'-CS was truncated by a nested transposable element, IS5564::ISPa157. The structure was even more complex since IS6100-ΔIS6100 structure and a TnAs2-like harbouring the operon merRTPADE was found downstream In1992. Fragments of TnAs3 harbouring 25-bp imperfect inverted repeats were identified bordering the intl1 of In1992 and also flanking IS6100-ΔIS6100, which might be genetic marks of its previous presence in the genome. Interestingly, In1992 also shows a distinct cassette array from In581 (blaGES-16-dfrA22-aacA27-aadA1), which was previously reported in Serratia marcescens strains recovered in Brazil. Finally, exoU gene, which encodes a potent cytotoxin of type III secretion systems (T3SS) effector proteins from P. aeruginosa and is associated to severe infections, was also detected. CONCLUSION: We described the novel In1992 carrying blaGES-16 surrounded by complex transposition events in a XDR P. aeruginosa strain. The identification of many sets of direct repeats adjacent to TnAs3 fragments indicates a major past of transposition events that shaped the current genetic environment of In1992.

Phenotypic and Molecular Characterization of Nonfermenting Gram-Negative Bacilli Causing Peritonitis in Peritoneal Dialysis Patients.

(1) Background: Peritonitis due to nonfermenting Gram-negative bacilli (NF-GNB) is a dramatic complication of peritoneal dialysis (PD) with bad outcomes. Previous studies of PD-related peritonitis due to Pseudomonas species have shown a low-resolution rate, without a high resistance rate to antipseudomonal antibiotics. This suggests that bacterial virulence factors can act and influence peritonitis evolution. This study aimed to describe the microbiological characteristics of NF-GNB causing PD-related peritonitis and analyze their influence on the outcome. (2) Methods: We analyze the 48 isolates from NF-GNB peritonitis, which were stored in our culture collection regarding bacterial resistance, biofilm, and other virulence factors' production, and clonal profile. Additionally, we collected data on treatment and outcomes from patients' clinical registers. (3) Results: The etiologies were species of Pseudomonas (50%), Acinetobacter (36%), and other NF-GNB (14%). There was a high (75%) proportion of biofilm producer lineages. The in vitro susceptibility rate of Pseudomonas spp. to amikacin, ciprofloxacin, and ceftazidime was significantly greater than that of Acinetobacter spp. and other species; however, there was a similar low-resolution rate (<45%) among the episodes attributable to them. Pseudomonas species have a polyclonal profile, while we found a clone of five multiresistant Acinetobacter baumannii over an 8-year interval (2000-2008), which suggest an origin from the healthcare environment. (4) Conclusions: We are not able to identify any predictor of outcome, but it is possible that biofilm and others virulence factors can act in concert and contribute to the bad outcome.

Identification of genus Acinetobacter: Standardization of in-house PCR and its comparison with conventional phenotypic methods.

BACKGROUND: Acinetobacter is grouped under nonfermenting Gram-negative bacilli. It is increasingly isolated from pathological samples. The ability of this genus to acquire drug resistance and spread in the hospital settings is posing a grave problem in healthcare. Specific treatment protocols are advocated for Acinetobacter infections. Hence, rapid identification and drug susceptibility profiling are critical in the management of these infections. AIMS: To standardize an in-house polymerase chain reaction (PCR) for identification of genus Acinetobacter and to compare PCR with two protocols for its phenotypic identification. METHODOLOGY: A total of 96 clinical isolates of Acinetobacter were included in the study. An in-house PCR for genus level identification of Acinetobacter was standardized. All the isolates were phenotypically identified by two protocols. The results of PCR and phenotypic identification protocols were compared. RESULTS: The in-house PCR standardized was highly sensitive and specific for the genus Acinetobacter. There was 100% agreement between the phenotypic and molecular identification of the genus. The preliminary identification tests routinely used in clinical laboratories were also in complete agreement with phenotypic and molecular identification. CONCLUSION: The in-house PCR for genus level identification is specific and sensitive. However, it may not be essential for routine identification as the preliminary phenotypic identification tests used in the clinical laboratory reliably identify the genus Acinetobacter.

Detection of blaVIM-7 in an extensively drug-resistant Pseudomonas aeruginosa isolate belonging to ST1284 in Brazil.

We described for the first time an extensively drug-resistant Pseudomonas aeruginosa isolate belonging to ST1284 carrying a plasmid-mediated blaVIM-7 in Brazil. The blaVIM-7 was harbored by an integron that also carried aacA4 and blaOXA-46. Multiple virulence factors were also detected.

Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry for species identification of nonfermenting Gram-negative bacilli.

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to identify 396 Nonfermenting Gram-Negative Bacilli clinical isolates was evaluated in comparison with conventional phenotypic tests and/or molecular methods. MALDI-TOF MS identified to species level 256 isolates and to genus or complex level 112 isolates. It identified 29 genera including uncommon species.

Nonfermenting Gram-negative Bacilli other than Pseudomonas aeruginosa and Acinetobacter Spp. Causing Respiratory Tract Infections in a Tertiary Care Center.

BACKGROUND: Nonfermenting gram-negative bacilli have emerged as important healthcare-associated pathogens. It is important to correctly identify all clinically significant nonfermenting gram-negative bacilli considering the intrinsic multidrug resistance exhibited by these bacteria. MATERIALS AND METHODS: A retrospective study was undertaken to identify the various nonfermenting gram-negative bacilli other than Pseudomonas aeruginosa and Acinetobacter spp. isolated from respiratory samples (n = 9363), to understand their clinical relevance and to analyze their antibiotic susceptibility pattern. RESULTS: Nonfermenting gram-negative bacilli were isolated from 830 (16.4%) samples showing significant growth. Thirty-three (4%) isolates constituted nonfermenting gram-negative bacilli other than P. aeruginosa and Acinetobacter spp. Stenotrophomonas maltophilia (15, 45.5%) was the most common isolate followed by Burkholderia cepacia (4, 12.1%), Sphingomonas paucimobilis (3, 9.1%), and Achromobacter xylosoxidans (3, 9.1%). On the basis of clinicomicrobiological correlation, pathogenicity was observed in 69.7% (n = 23) isolates. Timely and correct treatment resulted in clinical improvement in 87.9% cases. CONCLUSION: Any nonfermenting gram-negative bacilli isolated from respiratory tract infection should not be ignored as mere contaminant, but correlated clinically for its pathogenic potential and identified using standard methods so as to institute appropriate and timely antibiotic coverage.

Perioperative bacterial infections in deceased donor and living donor liver transplant recipients.

BACKGROUND: Deceased donor (DDLT) and living donor (LDLT) liver transplant (LT) is in vogue in several centers in India. Most centers are resorting to LDLT as a preferred surgery due to shortage of deceased donor liver. The risk of infection and its effect on survival in both groups of recipients from the Indian subcontinent are not known. The study was conducted to compare the bacterial infection rates among LDLT and DDLT recipients and their impact on survival at a tertiary referral center. METHODS: Retrospective data on 67 LT recipients were reviewed. Data on pre-, per-, and postoperative bacterial infection rates and the common isolates were obtained. RESULTS: Thirty-five patients had LDLT and 32 had DDLT. The prevalence of pre-operative bacterial infection and the isolates was similar in both groups. The perioperative bacterial infection rates were significantly higher in DDLT recipients (P < 0.01) (relative risk: 1.44 95% confidence interval 1.04-1.9). In both LDLT and DDLT, the common source was urinary tract followed by bloodstream infection. The common bacterial isolates in either transplant were Klebsiella followed by Escherichia coli, Pseudomonas spp. and nonfermenting gram-negative bacteria. Six patients (four LDLT; two DDLT) were treated for tuberculosis. Among the risk factors, cold ischemic time, and duration of stay in the intensive care unit was significantly higher for DDLT (p < 0.01). The death rates were not significantly different in the two groups. However, the odds for death were significantly high at 26.8 (p < 0.05) for postoperative bacterial infection and 1.8 (p < 0.001) for past alcohol. CONCLUSION: Liver transplant recipients are at high-risk for bacterial infection irrespective of type of transplant, more so in DDLT.

Analysis of influencing factors of mechanical ventilation time in esophageal cancer patients with lung infection after operation / 中国基层医药

ObjectiveTo explore the influencing factors of mechanical ventilation time in esophageal cancer patients with lung infection after operation.Methods55 esophageal cancer patients with lung infection after operation requiring endotracheal intubation or tracheostomy and mechanical ventilation were included.According to duration of mechanical ventilation time, ＜ 7 days for early weaned from ventilator group, ＞ 7 days for late weaning group.60 esophageal cancer patients with lung infection after operation without mechanical ventilation served as controls.Various factors on the impact of mechanical ventilation were compared.ResultsEarly and late weaning patients were older than control group(P ＜ 0.05), particularly in late weaning patients(P ＜ 0.01).Compared with the control, FEV1 and MVV decreased significantly in early weaning patients(P ＜ 0.05).NFGNB infection in late weaning patients significantly increased than in early weaning patients and the control(all P ＜ 0.01).Logistic multivariate analysis showed that FEV1 was independent factors affecting early weaning (P ＜ 0.01), age (P ＜ 0.05) and NFGNB infection (P ＜0.01) affecting late weaning.ConclusionThe influencing factors of mechanical ventilation time in esophageal cancer patiens with lung infection after operation were FEV1 decreasing,age and NFGNB infection.

Changes of Pathogens and Antimicrobial Resistance of Nonfermenting Gram-negative Bacilli in Intensive Care Unit / 中华医院感染学杂志

OBJECTIVE To study the isolation status and antimicrobial resistance of nonfermenting Gram-negative bacilli collected from intensive care unit(ICU) of our hospital so as to instruct the rational clinical application of antibiotics.METHODS The antimicrobial resistance of nonfermenting Gram-negative bacilli isolates collected from patients in ICU from Jan 2003 to Dec 2007 was analyzed.Antimicrobial susceptibility of clinical isolates were tested by Kirby-Bauer method.RESULTS Total 384 nonfermenting Gram-negative bacilli isolates were collected in 5 years.The most common species were Acinetobacter baumannii(219),Pseudomonas aeruginosa(117) and Stenotrophomonas maltophilla(36).The antimicrobial resistance rate of nonfermenting Gram-negative bacterial to most antibiotics were much higher.The antimicrobial resistance rate of Acinetobacter spp to imipenem,cefoperazone/sulbactam and piperacillin/tazobactam was 3.7%,28.3% and 42.9%.But the resistance rate of Acinetobacter spp to imipenem was increased in recent 2 years(58.0%).The antimicrobial resistance rate of P.aeruginosa to cefoperazone/sulbactam was the lowest.That of imipenem-resistant P.aeruginosa to cefoperazone/sulbactam was 34.0%.S.maltophilla was relatively susceptible to ceftazidime,cefoperazone/sulbactam and piperacillin/tazobactam.CONCLUSIONS Nonfermenters Gram-negative bacilli are the important pathogens in ICU.Surveillance of their prevalence and drug resistance may provide evidences for rational antibiotic choices.

Diversity of Antimicrobial Resistance among Gram-negative bacilli / 中华医院感染学杂志

OBJECTIVE To study the drug-resistant diversity of Gram-negative bacilli isolated from inpatients during recent five years.METHODS A total of 1 464 Gram-negative bacilli isolated were detected and retrospectively analyzed from 1999 to 2003.Antimicrobial susceptibility testing was performed using Kirby-Bauer method.RESULTS The resistance of Pseudomonas aeruginosa to piperacillin rised from 17.6% of 1999 to 79.2% of 2003,and that to ciprofloxacin rised from 4.3% of 1999 to 36.0% of 2003.The resistance of Escherichia coli to quinolones was above 50%,while to third-generation cephalosporins was 30-40%;the resistance of E.coli to piperacillin rised from 42.9% of 1999 to 68.9% of 2003,and that to ciprofloxacin rised from 40.0% of 1999 to 73.5% of 2003.The resistance of Acinetobacter to piperacillin rised from 31.2% of 1999 to 67.5% of 2003,and that to ceftriaxone rised from 36.0% of 1999 to 74.1% of 2003.The resistance of Serratia to ceftazidime,ceftriaxone,gentamicin,amikacin and piperacillin rised sharply.Imipenem was the most active antibiotic tested against Gram-negative bacilli.Cefoperazone/sulbactam and piperacillin/tazobactam also showed excellent activity against Gram-negative bacilli.CONCLUSIONS During recent five years,the resistance of the most common Gram-negative bacilli has increased rapidly.How to delay the resistance development of common strains become a global problem.