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A practical molecular identification of nonfermenting Gram-negative bacteria from cystic fibrosis

Identification of nonfermenting Gram-negative bacteria (NFGNB) of cystic fibrosis patients is hard and misidentification could affect clinical outcome. This study aimed to propose a scheme using polymerase chain reaction to identify NFGNB. This scheme leads to reliable identification within 3 days in an economically viable manner when compared to other methods.

Chronic respiratory tract infection is responsible for high morbidity and mortality in cystic fibrosis (CF) patients and is frequently associated with nonfermenting Gram-negative bacteria (NFGNB). 1 The microbiology of CF lung disease has changed substantially in recent decades, and now includes novel NFGNB such as Burkholderia cepacia complex (Bcc), Achromobacter xylosoxidans and Stenotrophomonas maltophilia, as well as several species of Ralstonia, Cupriavidus and Pandoraea. 1,2 Most exhibit high resistance to antimicrobials, which makes treatment problematic, and have the potential for interpatient transmission, leading some healthcare facilities to strongly recommend patient segregation. 1,3,4 Chronic infections, especially by Bcc species, may result in accelerated decline of pulmonary function. 5 This complex comprises at least 20 species, and although most are potentially capable of causing infections, Burkholderia cenocepacia and Burkholderia multivorans are considered the most prevalent, and B. cenocepacia is related to highly transmissible and virulent clonal lineages. 6 Some are associated with cepacia syndrome, a necrotizing pneumonia that leads to rapid deterioration of lung function, bacteremia and increased mortality. 5,7,8 A. xylosoxidans, which is considered the most common species within the genus Achromobacter, has been shown to cause a level of inflammation similar to Pseudomonas aeruginosa in chronically infected CF patients and a greater decline in lung function in these patients compared to non-infected patients. 9 Chronic pulmonary infection with S. maltophilia has been shown to be associated with an increased risk of pulmonary exacerbations, which leads to increased risk of lung transplantation or death in individuals with CF. 10 The large number of pili/fimbriae synthesized by S. maltophilia, which are associated with adhesion and biofilm formation, may contribute to the maintenance of this bacteria in lung infections, which shows why this microorganism is persistent and difficult to eradicate. 11 Since the consequence of these pathogens in the CF lung can be very serious, their correct identification is extremely important for a more efficient treatment.
Due to taxonomic complexity and high phenotypic similarity between these NFGNB, accurate identification represents a challenge for conventional microbiology. Conventional phenotypic methods including observation of colony morphology on media, analysis of manual biochemical reactions, and the use of automated and nonautomated commercially available biochemical panels are not suitable for CF isolates identification. NFGNB often present colonies of atypical appearance and lack key metabolic characteristics, which impairs the identification. 12,13 Automated systems, as Vitek ® 2 (bioMérieux), lead to inaccurate identification of NFGNB due to their phenotypic variations and slower growth rates. 14 Moreover, commercial phenotypic databases are often outdated and lack current taxonomy. 12 Misidentification of NFGNB seriously compromises infection control measures and confounds efforts to more clearly understand the epidemiology and natural history of infection in CF. 4,15 Currently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used in clinical microbiology laboratories for identification of CF bacterial species. It presents low cost per sample, and is considered to be a faster, and more reliable alternative than polymerase chain reaction (PCR) for these microorganisms identification. 15 However, the high cost of the equipment may be considered a limitation for some laboratories. 16 In addition, many recent studies report difficulty in identifying microorganisms at the species level, due to great variations of protein spectra in different strains belonging to the same species. [17][18][19] PCR is considered a simple and highly sensitive technique that produces results quickly, and it is economically viable when compared to sequencing methods. 20 The objective of the present study was to propose a scheme using PCR to identify NFGNB, based on the results of identification of reference strains and clinical isolates from CF patients.
The  Table 1 in order to evaluate the specificity of PCR assays.
The extraction of genomic DNA was performed using the method described by Gianni-Rossolini. 21 PCR was conducted in a volume of 25 L adding 2.5 L of 10× concentrated PCR buffer solution, 2 mM magnesium chloride (MgCl 2 ), 0.625 U Taq DNA polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.2 mM each of the 4 nucleotides (dNTP -Eppendorf, Hamburg, Germany), 20 pmol of the primers for amplification of genes, 60 ng of DNA and ultrapure water (Sigma-Aldrich, St. Louis, Missouri, USA). Amplification was carried out with the Mastercycler Gradient Thermal Cycler (Eppendorf, Hamburg, Germany). Cycling conditions for amplification were used according to the referenced articles (Table 1).
PCR was first performed according to Table 1. Modifications were proposed when nonspecific reactions were obtained or products were not amplified for the purpose of each primer ( Table 2).
This study included 201 clinical isolates of NFGNB previously identified by the Vitek ® 2 Compact system (bioMérieux, Marcy l'Etoile, France). The isolates were obtained from July  ; and production of oxidase, as previously described. 31 Identification of clinical isolates was performed by PCR using primers of genus/species according to proposed modifications by this study ( Table 2). All clinical isolates identified as belonging to Bcc were selected to perform restriction fragment length polymorphism analysis (PCR-RFLP) in order to identify the species of Bcc. Amplicons generated by PCR using the primers BCR1 and BCR2 were digested with HaeIII restriction endonuclease in accordance with the manufacturer's instructions (Thermo Fisher Scientific, Waltham, Massachusetts, USA). 32 Generated restriction fragments were analyzed by agarose gel electrophoresis (2%). Molecular size markers were used (100 bp ladder, Thermo Fisher Scientific, Waltham, Massachusetts, USA). After electrophoresis, the gels were stained with ethidium bromide and visualized and photographed with the AlphaImager System ® (Alpha Innotech, San Leandro, California, USA). All reference strains of Bcc were used as positive controls.
PCRs carried out for primers as shown in Table 2 allowed the recognition only of the genus for most NFGNB, except for primer pair SM-1/SM-4, which was specific to the genus and species for S. maltophilia.
According to Liu et al., 26 the primer pair used to identify A. xylosoxidans (AX-F1/AX-B1) showed 97% specificity. In this study, this primer pair generated amplicons only for Achromobacter genus. However, it generated amplicons for A. xylosoxidans, and for all isolates of A. piechaudii, A. denitrificans, A. dolens, A. insuavis, A. mucicolens, and A. ruhlandii (Table 3). According to Coenye et al., 28 the primer pair spuF/spuR was effective for identification of P. sputorum and genomospecies 2 and 3. In the present study, these primers generated amplicons for all species of Pandoraea tested, but they also generated amplicons for Ralstonia sp. The primer pair RalGS-F/RalGS-R provided amplicons for Ralstonia sp., but they also generated amplicons for Cupriavidus sp. The primer pair ral2f/ral2r, which is specific for Cupriavidus, also provided amplicons for three other bacteria genera: Achromobacter, Pandoraea and Ralstonia (Table 3). It was necessary to perform PCR using both primer pairs spuF/SpuR and RalGS-F/RalGS-R to allow identification of these microorganisms. If both primer pairs generate amplicons, the isolate should be identified as belonging to the genus Ralstonia. If there are amplicons only with the pair of primers spuF/spuR, the isolate should be identified as belonging to the Pandoraea genus. If after conducting PCR for the genera Pandoraea and Ralstonia, only amplicons from the primer pair RalGS-F/RalGS-R are generated, the Cupriavidus genus is suspected. Therefore, it is also necessary to obtain amplicons with primer pair ral2f/ral2r to confirm the identification as Cupriavidus.
Amplification with specific primers for bacterial species from Bcc did not produce nonspecific reactions to other bacterial genera that were not from Bcc. Primers used to identify the All PCRs were repeated at least five times to ensure reproducibility under the conditions described by the authors (Table 1) and under the proposed conditions for annealing temperature ( Table 2).
Analysis of PCR-RFLP patterns generated by digestion with the restriction enzyme HaeIII was able to discriminate all genomovars, except genomovars I and IIIA, which were differentiated by PCR. This analysis was also able to differentiate subgroups IIIA and IIIB of B. cenocepacia. Due to the high cost of sequencing, PCR-RFLP may be considered an option for clinical laboratories, especially because of its ability to identify the species B. multivorans and B. cenocepacia, which are linked to worse clinical prognoses than other species. 4  Based on the results of the identification of reference strains and clinical isolates, the present study proposed a PCR scheme for identification of emerging NFGNB isolated from CF patients (Fig. 1). The proposed scheme suggested that the first primers to be tested must have the highest specificity and first target the most frequent NFGNB with greater clinical impact.

Nonfer menting
The first step proposed was using the primer pairs BCR1/BCR2, AX-F1/AX-F2 and SM1/SM2 for Bcc, Achromobacter sp. and S. maltophilia, respectively. Within a shorter time frame compared with traditional tests, this approach allows the adoption of a segregation policy for individuals infected with Bcc. When amplification for Bcc occurs, PCR-RFLP and PCR with the primers BCRG3A1/BCRG3A2, which is used to identify B. cenocepacia (IIIA), should be performed to identify Bcc genomovars, since only PCR-RFLP was not able to discriminate B. cepacia from B. cenocepacia (IIIA).
The second step proposed was using the primer pairs spuF/spuR, RalGS-F/RalGSR and ral2f/ral2r to differentiate Pandoraea, Ralstonia and Cupriavidus, which were effectively identified by this scheme.
This scheme leads to reliable identification of all these microorganisms within three days, and this rapid identification allows for early antibacterial therapy and segregation. Comparative outcome studies are needed before conclusions about the relative virulence of specific strains can be drawn, and the first step is accurate identification in the laboratory. Furthermore, this scheme is more economically viable when compared to other methods based on sequencing. Therefore, this scheme can provide early bacterial diagnosis in CF patients, which can assist in increasing life expectancy.