Identification Of Nonfermenting Gram-negative Bacilli Research Articles

Prevalence and Identification of Nonfermentative Gram-negative Bacilli among Patients at Tertiary Care Hospital, Jaipur

Prevalence and Identification of Nonfermentative Gram-negative Bacilli among Patients at Tertiary Care Hospital, Jaipur

The Role of Matrix-Assisted Laser Desorption Ionization-Time of Flight- Mass Spectrometry (MALDI-TOF-MS) in the Identification of Non-fermenting Gram-Negative Bacilli from Clinical Isolates in a Tertiary Care Hospital

Non-fermenting Gram-negative bacilli (NFGNB) are a heterogeneous group of aerobic non-spore forming bacilli. Matrix-Assisted Laser Desorption Ionization-Time of Flight- Mass Spectrometry (MALDI-TOF-MS) is a quick, precise diagnostic method that could identify the isolates at the species level. The aim of the study is to identify the non-fermenters isolated from various clinical infections by MALDI-TOF analysis and to detect the antibiogram of these isolates. The non-fermenters were isolated from various clinical specimens, plated on Blood agar and MacConkey agar and incubated at 37⁰C for 24 hours. Identification was done on the basis of colony morphology, Gram stain, and MALDI-TOF Analysis. Antimicrobial Susceptibility Testing was done by Kirby- Bauer Disc Diffusion Technique using commercially available antibiotics on Muller-Hinton Agar. A total of 360 samples were studied. The majority of the non-fermenters were from Blood/ Body fluids (39.72%). Acinetobacter spp (31.67%) was the most common isolate, followed by Pseudomonas spp (22.50%). Other non-fermenters like Stenotrophomonas maltophilia, Burkholderia spp, Elizabethkingia spp, Achromobacter spp, Chryseobacterium spp, Ralstonia spp, Pandoraea spp, Rhizobium radiobacter, Sphingomonas spp, Ochrobactrum spp, Myroides odoratimimus and Brevundimonas spp were rarely isolated non-fermenters. Most of the strains were multi drug resistant to commonly used antibiotics like Cephalosporins, Aminoglycosides and Carbapenems. Our results showed that identifying non-fermenters using MALDI-TOF MS is a reliable and rapid technique that shortens the time to initiate appropriate therapy and even the length of the hospital stay.

Achieving pre-eminence of antimicrobial resistance among non-fermenting Gram-negative bacilli causing septicemia in intensive care units: A single center study of a tertiary care hospital.

Bloodstream infections in the intensive care unit have always been a global healthcare challenge. The present study was conducted with the aim to evaluate the yearly trend of antibiotic resistance in non-fermenting Gram-negative bacilli (NFGNB) causing septicemia in intensive care units. Blood samples were collected from the patients admitted in various intensive care units and processed for isolation and identification of non-fermenting Gram-negative bacilli. The isolated bacterial strains were subjected to antibiotic susceptibility testing as per standard operating procedures. Out of 3632 blood samples, 977 (26.9%) samples showed microbial growth, of which 10.1% were Gram positive cocci, 8.7% were Gram negative bacilli (Enterobacterales), 7% were NFGNB and 1% were Candida spp. Increasing resistance among Acinetobacter baumannii complex was observed to ceftazidime, cefepime, amikacin, ciprofloxacin, levofloxacin, meropenem and trimethoprim-sulfamethoxazole. Moreover, Pseudomonas aeruginosa strains were found to be associated with increased resistance to ciprofloxacin, levofloxacin, ceftazidime and meropenem. A substantial increase in resistance levels was observed among Stenotrophomonas maltophilia and Sphingomonas paucimobilis as well. An increasing trend of antimicrobial resistance in NFGNB envisages the worst consequences in ICUs in the coming years. Therefore, reviewing and strict implementation of the antimicrobial policies including 'rational use of antibiotics' is recommended.

Non-fermenting gram negative bacilli isolated from blood and their in vitro sensitivity pattern in a tertiary care hospital

Background: Bacteremia caused by non-fermenting gram negative bacilli (NFGNB) is a serious infection which is associated with high morbidity and mortality. Identification of NFGNB and monitoring their susceptibility pattern are important for proper management of these infections. Aim: Scope of the present study is to characterize the NFGNB along with their antimicrobial sensitivity pattern among the patients coming to our hospital, a tertiary care center. Materials & Methods: A retrospective study was conducted to determine the NFGNB isolated from blood during the one-year study period. A total of 8223 Blood cultures were received in the laboratory which was processed by semi-automated technique in Bac-T Alert. Antibiotic susceptibility was performed by VITEK and Kirby Bauer methods and interpreted by CLSI guidelines. 102 isolates of NFGNB were obtained from the Blood culture. Results: Most commonly isolated organism is Acinetobacter species (47%) followed by Pseudomonas spp (27%) and Burkholderia spp(15.6%). Maximum isolates of Acinetobacter spp were sensitive to colistin (91.9%), cefoperazone-sulbactum (88.4%), Tigecycline (62.5%). Pseudomonas spp was sensitive to colistin (100%), ciprofloxacin (59%). Conclusion: Identification of NFGNB and monitoring their susceptibility patterns will help in proper management of infections caused by them.

Quality of dialysis water and dialysate in haemodialysis centres: Highlight for occurrence of non-fermenting gram-negative bacilli.

To evaluate the physicochemical and microbiological quality of dialysis water and dialysate samples from haemodialysis centres. Samples were fortnightly collected from three haemodialysis centres in Bauru City, Brazil, between July 2017 and June 2018, at the stages of post-reverse osmosis, reuse and dialysate. Analyses included determination of conductivity, fluoride, nitrate and sulphate; test for total coliform bacteria; count of heterotrophic bacteria; count and identification of non-fermenting gram-negative bacilli (NFGNB); drug susceptibility test; biofilm formation capacity; and genetic similarity among some isolated NFGNB. Of the analysed samples, only 4/72 (5.6%) had conductivity values ≥10 mS/cm, 4/216 (1.9%) presented total coliforms and 1/216 (0.5%) had heterotrophic bacteria count >100CFU/ml. NFGNB were isolated from 99/216 (45.8%) samples, and the major identified micro-organisms included Herbaspirillum aquaticum/huttiense, Brevundimonas aurantiaca, Cupriavidus metallidurans, Pseudomonas aeruginosa and Ralstonia insidiosa. Isolates of P. aeruginosa and Burkholderia cepacia complex were sensitive to most antimicrobials and, together with isolates of Ralstonia insidiosa and Ralstonia pickettii, showed strong biofilm formation capacity. Some isolates expressed the same electrophoretic profile on pulsed-field gel electrophoresis, indicating the persistence of bacterial clones in the systems over time. NFGNB were observed in several dialysis water and dialysate samples from all investigated centres, which may represent a risk to the health of patients. Regular inclusion of actions for NFGNB control and monitoring in haemodialysis fluids are suggested for greater safety of the dialytic process.

Non-Fermenting Gram Negative Bacteria as Uropathogens in Causing Urinary Tract Infection and its Antimicrobial Susceptibility Pattern at A Tertiary Care Centre of South India

Non fermenting gram-negative bacilli (NFGNB) are recently striving as uropathogens. The present study was conducted to isolate the common species of bacteria in NFGNB causing urinary tract infection (UTI) and its correlation with comorbid conditions and to study the antibacterial susceptibility pattern. This retrospective study was done at the diagnostic Microbiology laboratory of a tertiary care hospital. Urine samples were collected for the period of six months. These samples were plated on blood agar and MacConkey agar and incubated at 37°C for 18–24 hr under aerobic conditions. Identification of NFGNB was done by Gram staining and MALDI-TOF (Matrix- Assisted Laser Desorption/ Ionization- Time of Flight, Biomerieux- Diagnostics). Antibiotic sensitivity testing was done by Vitek® 2 system (Biomerieux- Diagnostics) using N 281 card. Data was analyzed using SPSS IBM version 16. Out of the total 16,413 non repetitive urine samples that were received in the laboratory, 318 had significant bacteriuria. NFGNB were identified in 108 (33.9%) of all the urine samples with significant bacteriuria. Prevalence of non-fermenters in our study was 0.6%. NFGNB were more frequently isolated in the females and also in the age group of more than 50 years. Eighty five (78.70%) had comorbid conditions. P. aeruginosa and A. baumannii were the most common organism isolated among NFGNB. Pseudomonas aeruginosa isolates showed high susceptibility to imipenem (80.2%) and amikacin (66.6%). NFGNB although seen frequently in females and in age group of 50 years and above, clinical correlation with comorbid condition is essential to label it as uropathogens. Amikacin or imipenem may be the empirical drug of choice.

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy for Rapid Identification of Non-Fermenting Gram-Negative Bacilli Isolated from Patients with Cystic Fibrosis

BackgroundChronic respiratory infections with non-fermenting Gram-negative bacilli are a key feature of cystic fibrosis (CF). For microbiology laboratories, rapid and accurate identification of these bacteria is often challenging and labor intensive. This study was undertaken to evaluate whether attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy could rapidly discriminate Pseudomonas aeruginosa (mucoid and non-mucoid), Burkholderia cepacia complex, Burkholderia gladioli, Achromobacter spp. and Stenotrophomonas maltophilia.MethodsA total of 263 well-characterized clinical strains isolated from respiratory samples of patients with CF attending the CHU Sainte-Justine CF clinic were included in this study, consisting of 70 P. aeruginosa, 83 Burkholderia spp., 52 Achromobacter spp. and 58 Stenotrophomonas maltophilia isolates from the biobank. Isolates were thawed and sub-cultured twice on sheep blood (5%) agar. ATR-FTIR spectral acquisition was performed in triplicate for each isolate. Multivariate statistical analysis of the ATR-FTIR spectra was performed by hierarchical cluster analysis (HCA) and principal component analysis (PCA) in conjunction with the use of a feature selection algorithm.ResultsAn ATR-FTIR spectral database consisting of 789 spectra of P. aeruginosa, Burkholderia spp., Achromobacter spp. and Stenotrophomonas maltophilia was created in this study. Complete discrimination among all four genera as well as among three species within the B. cepacia complex and B. gladioli was achieved based on HCA and PCA of the spectra in the database. ATR-FTIR analysis of a validation set consisting of 30 isolates was conducted in parallel with identification by MALDI-TOF mass spectrometry and yielded >95% concordance between the two techniques.ConclusionATR-FTIR spectroscopy is a promising tool for rapid, inexpensive and accurate identification of non-fermenting Gram-negative bacilli. Additional work is needed to further expand the spectral database, particularly with mucoid strains.Disclosures All authors: No reported disclosures.

93 The use of MALDI-TOF MS for the identification of non-fermenting Gram-negative bacilli isolated from cystic fibrosis patients

93 The use of MALDI-TOF MS for the identification of non-fermenting Gram-negative bacilli isolated from cystic fibrosis patients

Antibiotic resistance profile of non-fermenting Gram-negative bacilli isolated from the blood cultures of cancer patients.

Sir, Non-fermenting Gram-negative bacilli (NFGNB) are a group of organisms that either do not utilize glucose as a source of energy or utilize it oxidatively. NFGNB can cause a significant number of infections, ranging from superficial to deep-seated and disseminated infections in immunocompromised hosts, neutropenic patients, patients with cystic fibrosis, patients on mechanical ventilation and indwelling catheters, and patients undergoing invasive diagnostic and therapeutic procedures. Blood stream infections (BSI) caused by NFGNB are a major cause of morbidity and mortality worldwide. High intrinsic resistance of NFGNB to antimicrobial compounds makes the treatment of BSIs caused by them difficult and expensive. NFGNB have a varied isolation rate, ranging from 2.18 to 45.9%, as observed from various clinical samples.[1] In an attempt to recognize the antibiotic resistance and prevalence pattern among NFGNB associated with BSIs in cancer patients, a prospective study was conducted over a period of one year from July 2013 to June 2014, at a tertiary care cancer institute in North India. Blood culture was performed in BacT/Alert blood culture bottles (bioMerieux, Durham, North Carolina, USA). From the positive bottles, the subcultures were performed on MacConkey agar and blood agar. The VITEK®2 Compact (C) system (bioMerieux, North Carolina, USA) was used to identify NFGNB up to the species level, using the Gram-negative Identification (GN-ID) 21341 card, and antibiotic susceptibility testing (AST) was done using the AST 281 card. The antibiotic susceptibility results were expressed as susceptible, intermediate or resistant, according to the criteria of the Clinical Laboratory Standards Institute (CLSI) M100-S23 (2013).[2] Among 815 total samples received for blood culture, 249 bottles were flagged as positive in the BacT Alert 3D. However, only 172 yielded pathogenic bacteria on subculture. Sixty-three (36.6%) were Gram-negative and 108 (62.7%) were Gram-positive organisms, while one isolate was fungus (Candida sp.). Forty, out of 63 (63.5%) Gram-negative organisms were NFGNB, out of which Burkholderia cepacia (60%) was the predominant isolate. Acinetobacter baumannii (20%), Pseudomonas aeruginosa (12.5%), Pseudomonas stutzeri (2.5%), Alcaligenes faecalis (2.5%), and Sphingomonas paucimobilis (2.5%) were the other NFGNB isolated from the blood stream infections. The antibiotic susceptibility results are given in Table 1, which shows the percentage of resistant isolates. A high level of resistance against the first- and second-line antibiotics was noted. In this study, NFGNB were isolated from 4.9% of the total blood samples received for the culture. Out of the total Gram-negative bacilli, the NFGNB in our study were 63.5% (40/63), and B. cepacia was the predominant NFGNB. Prabash et al., in 2010, analyzed the blood cultures of cancer patients (neutropenic and non-neutropenic) and found that non-fermenters comprised a majority of the Gram-negative bacilli isolated (62.24%), with Pseudomonas (30.37%) and Acinetobacter sp. (11.57%) being the predominant NFGNBs.[3]Table 1: Antibiotic resistance pattern (percentage) of NFGNB isolated from blood cultureThe in vitro anti-microbial susceptibility of Burkholderia cepacia isolates revealed 92% resistance to meropenem, while the susceptibility to co-trimoxazole was 100%. The 2010 Sanford Guide recommends co-trimoxazole as the treatment of choice for these infections. The other antibiotics to which B. cepacia isolates are susceptible are meropenem, ceftazidime, ceftibuten, chloramphenicol, and trimethoprim.[4] However, according to literature, the antibiogram of B. cepacia isolates from cancer patients may be slightly different, with increased susceptibility to some antibiotics, especially piperacillin, piperacillin-tazobactam, and cefepime.[56] One hundred and 87.5% of A. baumannii isolates were resistant to aminoglycosides and colistin, respectively, while all the isolates were susceptible to meropenem. Among the Pseudomonas species, a high level of resistance was recorded for ceftazidime (50%), aztreonam (50%), and doripenem (50%). Colistin and cefepime showed maximum activity with an overall susceptibility of 100 and 83.3%, respectively. A single isolate of A. faecalis (2.5%) was resistant to all antibiotics, except amikacin. However, S. paucimobilis (2.5%) was a fairly sensitive strain, with resistance observed only against ceftazidime and aztreonam. The emerging challenges of multidrug resistance, both intrinsic and acquired, among NFGNB, are of serious concern to the treating physician. Improved antibiotic stewardship and infection control measures will be needed to prevent or slow down the emergence and spread of multidrug-resistant NFGNB in the healthcare setting. Identification of NFGNB and monitoring their susceptibility patterns will help in improving the empirical therapy.

Bronchiolitis and bacteraemia caused by Burkholderia gladioli in a non-lung transplantation patient.

Dear Editor, The Gram-negative bacillus Burkholderia gladioli is known to be a causative pathogen of pneumonia in cystic fibrosis 1,2. Chronic lower respiratory infections involving B. gladioli are associated with poor graft survival and unfavourable outcomes 1,2. Burkholderia gladioli was first identified as a plant pathogen in gladiolus, iris and rice, but this bacterium is found in diverse environments including soil, plants and the human respiratory tract. Its pathogenesis in humans was first described in the mid-1990s in case reports of cystic fibrosis 3 and chronic granulomatous disease 4, and the majority of the reported B. gladioli infections have been in immunocompromised adults, e.g. human immunodeficiency virus-infected patients, and newborns 5. Although it is still a fairly uncommon pathogen, B. gladioli in humans is an opportunistic pathogen and is associated with a poor prognosis. The genus Burkholderia was formerly classified as the genus Pseudomonas. The virulence of B. gladioli in humans is recognized to be low, and the clinical manifestations of this pathogen in non-lung transplantation patients have not been fully elucidated. We treated a 62-year-old female patient with myasthenia gravis, a thymoma and immune-mediated granulocytopenia. She had been treated with tacrolimus for the previous 12 months. From her sputum, non-fermenting Gram-negative rods were cultured microbiologically. The isolate was oxidase-negative, indole-negative and nitrate-negative. The 16S rRNA detection in our collaborator's laboratory confirmed B. gladioli as a 99% comparable DNA sequence. The patient's case was complicated with bronchiolitis followed by bacteraemia due to B. gladioli. The infection of B. gladioli presented as diffuse panbronchiolitis (Fig.​(Fig.1)1) and progressed into repeated bacteraemia. The pathogen was not isolated because a laboratory technician made an arbitrary decision not to proceed further to determine the species of the causative organism, although the patient had been suffering from repeated bacteraemia due to the same bacillus. Our institute's laboratory reported the pathogen only as ‘an environmental non-fermenting Gram-negative rod.’ Figure 1 Pulmonary infection by Burkholderia gladioli presenting as diffuse panbronchiolitis. (a) Transverse view showing the transbronchial distribution of small opaque nodules in the right upper lobe (S2), indicated by arrowheads. (b) Nodular infiltration shadows formed ... Non-fermenting Gram-negative bacilli are bradytrophic and can survive for long periods in a wet environment without sufficient nutrients. Such bacilli include Pseudomonas spp., Acinetobacter spp., Stenotrophomonas spp., Chryseobacterium spp. and Burkholderia spp., all of which are opportunistic pathogens. Non-fermenting Gram-negative bacilli have the following microbiological characteristics: (1) they produce various β-lactamases in nature, (2) they show multidrug resistance in nature, (3) they easily acquire plasmids harbouring antibiotic-resistant genes, and (4) they form a biofilm on surfaces to colonize. All of these features are linked to clinical issues in the treatment of infection, and they are linked to unfavourable outcomes. As stated in the position paper distributed by the US Health Care Infection Control Practices Advisory Committee (HICPAC), the surveillance of newly emerging pathogens is a critically important component of prudent infection control. Microbiological identification is also crucial, especially using samples obtained in clinical settings. In our patient, the isolated bacterium was not multidrug-resistant, fortunately; she was treated successfully with a carbapenem antibiotic, meropenem (0.5 g, three times per day, administered for 11 days). We recommend the microbiological identification of non-fermenting Gram-negative bacilli according to the clinical situation, even when the bacillus is likely to be found in the environment and is an unremarkable microbe. To do so, interdisciplinary interactions between clinical and laboratory personnel are helpful. The value of medical microbiology is based on interactive laboratories.

Prevalence of non-fermenting Gram-negative bacilli and their in vitro susceptibility pattern at a tertiary care teaching hospital

Background: Aerobic non-fermenting Gram-negative bacilli (NFGNB) once considered as contaminants now associated with life-threatening infections and emerging as multi drug resistant nosocomial pathogens. Aim: Isolation and identification of NFGNB in all the clinical samples and to determine antibiotic susceptibility pattern of the isolated NFGNB. Materials and Methods: This study has been conducted in the Department of Microbiology at a tertiary care teaching hospital over a period of 2 months from September to October 2013. NFGNB were isolated and identified from clinical specimens by standard procedure and antibiotic sensitivity test was performed. Results: NFGNB isolation rate in the present study was 3.58%. Male to female ratio was 2.125. Pus was the most common specimen (21%) followed by tracheal aspirate (17%). Pseudomonas aeruginosa was the most common isolate (60%) followed by Acinetobacter baumannii (22%) and Acinetobacter lwoffii (12%). P. aeruginosa has shown good sensitivity to amikacin (83.3%), imipenem (80%) and piperacillin-tazobactam (73.3%) whereas A. baumannii showed multidrug resistance. Conclusion: It is necessary to identify NFGNB and to monitor their susceptibility pattern to guide the clinician for better care and management of patients. NFGNB are now emerging as organisms of nosocomial infections. Hence, antibiotic sensitivity testing and infection control measures are needed to prevent the emergence and spread of multi drug resistant NFGNB in health care settings.

Prevalence of non-fermenting gram negative bacilli and their in vitro susceptibility pattern in a tertiary care hospital of Uttarakhand: A study from foothills of Himalayas

Introduction: Non-fermenting gram negative bacilli (NFGNB) are taxonomically diverse group of pathogens that has emerged as a major cause of health care associated infections especially in immunocompromised hosts. Identification of NFGNB and monitoring their susceptibility pattern are important for proper management of infections caused by them. Prevalence and antibiogram of NFGNB has not yet been reported from this part of India. Aim of the present study was to characterize the prevalence of NFGNB along with their antimicrobial sensitivity pattern among the patients coming to our hospital a tertiary care center. Materials and Methods: A total of 2585 various clinical specimens were received in laboratory and were processed. Non fermenters were identified using a standard protocol. Antimicrobial susceptibility testing was performed by Kirby Bauer disc diffusion method. Results: Among 2585 clinical samples 241 yielded NFGNB accounting for an isolation rate of 9.32% and a total of 246 non fermenters were grown as five samples yielded two types of NFGNB. Pseudomonas species (49.59%) and Acinetobacter species (43.09%) were the most commonly isolated NFGNB. A high level of antibiotic resistance was recorded for most of the first and second line drugs. Imipenem and amikacin were the drugs with maximum activity. Overall imipenem resistance was found to be 30.54%. Conclusion: Identification of NFGNB and monitoring their susceptibility patterns will help in proper management of infections caused by them. Improved antibiotic stewardship and infection-control measures will be needed to prevent or slow the emergence and spread of multidrug-resistant NFGNB in the healthcare setting.

Evaluation of the Bruker Biotyper and Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Systems for Identification of Nonfermenting Gram-Negative Bacilli Isolated from Cultures from Cystic Fibrosis Patients

The Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) instruments were evaluated for the identification of nonfermenting gram-negative bacilli (NFGNB) by a blinded comparison to conventional biochemical or molecular methods. Two hundred NFGNB that were recovered from cultures from cystic fibrosis patients in the University of Iowa Health Care (UIHC) Microbiology Laboratory between 1 January 2006 and 31 October 2010 were sent to Mayo Clinic for analysis with the Bruker Biotyper (software version 3.0) and to bioMérieux for testing with Vitek MS (SARAMIS database version 3.62). If two attempts at direct colony testing failed to provide an acceptable MALDI-TOF identification, an extraction procedure was performed. The MS identifications from both of these systems were provided to UIHC for comparison to the biochemical or molecular identification that had been reported in the patient record. Isolates with discordant results were analyzed by 16S rRNA gene sequencing at UIHC. After discrepancy testing, the Bruker Biotyper result agreed with the biochemical or molecular method, with 72.5% of isolates to the species level, 5.5% to the complex level, and 19% to the genus level (3% not identified). The level of agreement for Vitek MS was 80% species, 3.5% complex, 6% genus, and 3.5% family (7% not identified). Both MS systems provided rapid (≤3 min per isolate) and reliable identifications. The agreement of combined species/complex/genus-level identification with the reference method was higher for the Bruker Biotyper (97% versus 89.5%, P = 0.004) but required an extraction step more often. Species-level agreement with the reference method was similar for both MS systems (72.5% and 80%, P = 0.099).

Comparison of Traditional Phenotypic Identification Methods with Partial 5′ 16S rRNA Gene Sequencing for Species-Level Identification of Nonfermenting Gram-Negative Bacilli

Correct identification of nonfermenting Gram-negative bacilli (NFB) is crucial for patient management. We compared phenotypic identifications of 96 clinical NFB isolates with identifications obtained by 5' 16S rRNA gene sequencing. Sequencing identified 88 isolates (91.7%) with >99% similarity to a sequence from the assigned species; 61.5% of sequencing results were concordant with phenotypic results, indicating the usability of sequencing to identify NFB.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Nonfermenting Gram-Negative Bacilli Isolated from Cystic Fibrosis Patients

The identification of nonfermenting gram-negative bacilli isolated from cystic fibrosis (CF) patients is usually achieved by using phenotype-based techniques and eventually molecular tools. These techniques remain time-consuming, expensive, and technically demanding. We used a method based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the identification of these bacteria. A set of reference strains belonging to 58 species of clinically relevant nonfermenting gram-negative bacilli was used. To identify peaks discriminating between these various species, the profile of 10 isolated colonies obtained from 10 different passages was analyzed for each referenced strain. Conserved peaks with a relative intensity greater than 0.1 were retained. The spectra of 559 clinical isolates were then compared to that of each of the 58 reference strains as follows: 400 Pseudomonas aeruginosa, 54 Achromobacter xylosoxidans, 32 Stenotrophomonas maltophilia, 52 Burkholderia cepacia complex (BCC), 1 Burkholderia gladioli, 14 Ralstonia mannitolilytica, 2 Ralstonia pickettii, 1 Bordetella hinzii, 1 Inquilinus limosus, 1 Cupriavidus respiraculi, and 1 Burkholderia thailandensis. Using this database, 549 strains were correctly identified. Nine BCC strains and one R. mannnitolilytica strain were identified as belonging to the appropriate genus but not the correct species. We subsequently engineered BCC- and Ralstonia-specific databases using additional reference strains. Using these databases, correct identification for these species increased from 83 to 98% and from 94 to 100% of cases, respectively. Altogether, these data demonstrate that, in CF patients, MALDI-TOF-MS is a powerful tool for rapid identification of nonfermenting gram-negative bacilli.

16S rRNA Gene Sequencing versus the API 20 NE System and the VITEK 2 ID-GNB Card for Identification of Nonfermenting Gram-Negative Bacteria in the Clinical Laboratory

Over a period of 26 months, we have evaluated in a prospective fashion the use of 16S rRNA gene sequencing as a means of identifying clinically relevant isolates of nonfermenting gram-negative bacilli (non-Pseudomonas aeruginosa) in the microbiology laboratory. The study was designed to compare phenotypic with molecular identification. Results of molecular analyses were compared with two commercially available identification systems (API 20 NE, VITEK 2 fluorescent card; bioMérieux, Marcy l'Etoile, France). By 16S rRNA gene sequence analyses, 92% of the isolates were assigned to species level and 8% to genus level. Using API 20 NE, 54% of the isolates were assigned to species and 7% to genus level, and 39% of the isolates could not be discriminated at any taxonomic level. The respective numbers for VITEK 2 were 53%, 1%, and 46%, respectively. Fifteen percent and 43% of the isolates corresponded to species not included in the API 20 NE and VITEK 2 databases, respectively. We conclude that 16S rRNA gene sequencing is an effective means for the identification of clinically relevant nonfermenting gram-negative bacilli. Based on our experience, we propose an algorithm for proper identification of nonfermenting gram-negative bacilli in the diagnostic laboratory.

Caractéristiques phénotypiques et génotypiques des souches atypiques de bacilles à Gram négatif non fermentants isolées chez des patients atteints de mucoviscidose

We used partial 16S rRNA gene (16S DNA) sequencing for the prospective identification of nonfermenting Gram-negative bacilli recovered from patients attending our cystic fibrosis center (hôpital Necker–Enfants malades), which gave problematic results with conventional phenotypic tests. During 1999, we recovered 1093 isolates of nonfermenting Gram-negative bacilli from 702 sputum sampled from 148 patients. Forty-six of these isolates (27 patients) were not identified satisfactorily in routine laboratory tests. These isolates were identified by 16S DNA sequencing as Pseudomonas aeruginosa (19 isolates, 12 patients), Achromobacter xylosoxidans (10 isolates, 8 patients), Stenotrophomonas maltophilia (9 isolates, 9 patients), Burkholderia cepacia genomovar I/III (3 isolates, 3 patients), Burkholderia vietnamiensis (1 isolate), Burkholderia gladioli (1 isolate) and Ralstonia mannitolilytica (3 isolates, 2 patients). Fifteen isolates (33%) were resistant to all antibiotics in routine testing. Sixteen isolates (39%) resistant to colistin were recovered on B. cepacia-selective medium: 2 P. aeruginosa, 3 A. xylosoxidans, 3 S. maltophilia and the 8 Burkholderia - Ralstonia isolates. The API 20NE system gave no identification for 35 isolates and misidentified 11 isolates (2 P. aeruginosa, 2 A. xylosoxidans and 1 S. maltophilia classified as B. cepacia ). Control measures and/or treatment were clearly improved as a result of 16S DNA sequencing in three of these cases. This study confirms the weakness of phenotypic methods for identification of atypical nonfermenting Gram-negative bacilli recovered from cystic fibrosis patients. The genotypic methods, such as 16S DNA sequencing which allows identification of strains in routine practice, appears to have a small, but significant impact on the clinical management of CF patients.

Biotyping of Acinetobacter calcoaceticus using the API 2ONE system

The API 2ONE system for the identification of non-fermentative Gram-negative bacilli enables the discrimination of a possible 209 different biotypes of Acinetobacter spp. and consequently has potential for use as an Acinetobacter spp. typing system. A total of 122 separate strains of Acinetobacter spp. isolated in Nottingham hospitals over a 4 year period from a wide variety of clinical specimens, divided into 31 different biotypes which were stable over a 1 year storage period. Two biotypes predominated, one of which was a multiply resistant strain of A. calcoaceticus variant anitratus. The API 2ONE system was found to be a rapid method for biotyping strains of Acinetobacter spp. and was helpful in monitoring cross-infection and spread of particular strains of Acinetobacter spp. in the hospital environment.

Evaluation of the N/F® System for Identification of Nonfermentative Gram-Negative Bacilli Using a Reference Laboratory Population

The Corning N/F® system was evaluated for possible use in a reference laboratory by assessing the results obtained in testing 220 clinical isolates evenly distributed into 49 species or biotypes. The organisms chosen were indicative of the variety and distribution of nonfermentative gram-negative bacilli that are often received for identification by a reference laboratory, and included organisms not listed in the N/F data base. The complete N/F system consists of the two-tube N/F-Screen® and the Uni-N/F-Tek® plate. The two N/F-Screen tubes alone, intended to identify Pseudomonas aeruginosa and the Pseudomonas fluorescens/putida group, identified five of 17 of these isolates. The complete system correctly identified 55% of 220 isolates to the level of identification required by a reference laboratory. Of the 173 test organisms included in the N/F data base, 75% were identified to the level of identification listed by the manufacturer. However, the more commonly isolated organisms were identified with greater accuracy. In particular, all isolates of Acinetobacter calcoaceticus, Alcaligenes odorans, Moraxella osloensis, six species of Pseudomonas including Pseudomonas aeruginosa and four biotypes of Pseudomonaslike groups were correctly identified by the system.

Use of casein, tyrosine, and hypoxanthine in the identification of nonfermentative gram-negative bacilli.

Four hundred and five (405) strains of nonfermentative gram-negative bacilli (NFB) were identified using conventional tests. The NFB studied include 323 originating from clinical specimens, 37 from environmental samples, and 45 authentic strains from other workers and from our culture collections. All these strains were studied for their ability to degradate casein, tyrosine, and hypoxanthine. It was found that these tests are useful in identifying NFB within 24 to 48 h when used with other tests.