PCR Assay Based on the gyrB Gene for Rapid Identification of Acinetobacter baumannii‐calcoaceticus Complex at Specie Level

Abstract

Background

The genus Acinetobacter sp. comprises more than 50 species, and four are closely related and difficult to be distinguished by either phenotypic or genotypic methods: the Acinetobacter calcoaceticus‐baumannii complex (ABC). The correct identification at species level is necessary mainly due to the epidemiological aspects.

Methods

We evaluated a multiplex PCR for gyrB gene to identify the species of the ABC using the sequencing of the ITS 16S‐23S fragment as a gold standard. Isolates identified as Acinetobacter calcoaceticus‐baumannii from three hospitals at southern Brazil in 2011 were included in this study.

Results

A total of 117 isolates were obtained and 106 (90.6%) were confirmed as A. baumannii, 6 (5.1%) as A. nosocomialis and 4 (3.4%) as A. pittii by PCR for gyrB gene. Only one isolate did not present a product of the PCR for the gyrB gene; this isolate was identified as Acinetobacter genospecie 10 by sequencing of ITS. We also noted that the non‐A. baumannii isolates were recovered from respiratory tract (8/72.7%), blood (2/18.2%) and urine (1/9.1%), suggesting that these species can cause serious infection.

Conclusion

These findings evidenced that the multiplex PCR of the gyrB is a feasible and simple method to identify isolates of the ABC at the species level.

Introduction

Originally, only two species were described in the genus Acinetobacter: A. calcoaceticus and A. lwoffii. Later, Bouvet and Grimont defined 12 genospecies based in DNA‐DNA hybridization. Thereafter, new species have been described and, currently, the genus comprises 51 species with valid published names that were delineated by DNA‐DNA hybridization, 16S‐rRNA and whole genome sequencing among others 1, 2, 3, 4, 5.

Despite the fact that many identification methods have been described, there are four closely related species which are difficult to be distinguished by either phenotypic or genotypic methods. These were referred as Acinetobacter calcoaceticus‐ baumannii complex (ABC complex) and comprises groups 1, 2, 3 e 13 of Bouvet and Grimont (A. calcoaceticus, A. baumannii, A. genospecie 3 and A. genospecie 13TU, respectively) 2, 3. More recently, A. genospecie 3 and A. genospecie 13TU were named Acinetobacter pittii and Acinetobacter nosocomialis, respectively, according to a detailed study of their genotypic characteristics 4.

It is important to identify the species of the ABC complex as they may present distinct epidemiological and clinical contexts. The species A. baumannii, A. pittii and A. nosocomialis are found more frequently in clinical specimens and are related to nosocomial infections. A. baumannii is by far the specie most commonly involved in nosocomial infections and usually present resistance to multiple antibiotics (MDR). On the other hand, A. calcoaceticus is commonly obtained from soil and water and not observed in reports of diseases 5, 6, 7.

A. baumannii has already been widely described as a cause of nosocomial infections and is associated with high level of mortality and a poor outcome mainly because A. baumannii isolates from outbreaks usually are carbapenems resistant. In contrast, only a few reports have described carbapenem resistance among A. pittii and A. nosocomialis 8, 9, 10.

Several methodologies have been described to identify the ABC species. Commercial methods (automated and semi‐automated) of identification present low performance to distinguish the species as well as other phenotypic methods. Methodologies based in nucleic acids analysis (such as DNA‐DNA hybridization, amplified rRNA gene restriction analysis—ARDRA, tRNA spacer fingerprinting, partial sequencing of the rpoB gene and the 16S‐23S rRNA gene spacer region—ITS) have improved the identification of these species but these methods are rarely used in the routine of microbiology laboratories as they are too expensive 4, 11, 12, 13, 14.

The gyrB gene corresponds to the fragment encoding the β subunit of the enzyme DNA gyrase and along with the multilocus analysis studies it proved to be heterogeneous interspecies. Therefore, analysis of the gyrB gene is a promising tool to differentiate species of ABC complex due to its high specificity and ease of implementation. PCR products of the gyrB gene are of different sizes for each species, allowing the distinction among them. Higgins et al. (2010) proposed a multiplex PCR method with 7 primers targeting this gene which does not need DNA sequencing and, therefore, is a fast and less laborious method which would allow differentiation among the A. calcoaceticus and A. pittii (A. genomic species 3) 15.

In this study, we evaluated the multiplex PCR for gyrB gene to identify the species of the ABC complex using the 16S‐23S rRNA gene spacer region—ITS sequencing as the gold standard.

Materials and Methods

Bacterial isolates

A total of 118 isolates obtained from patients at three different hospitals in southern Brazil, during 2011, were included in this study. These isolates were identified initially as Acinetobacter calcoaceticus‐baumannii by Vitek 2 system^® (bioMeriéux, Marcy l'Etoile, France).

DNA preparation

The boiling method described by Vaneechoutte et al. (1995) was used to extract the DNA from the isolates. DNA was stored at −20°C until use.

Amplification 16S‐23S rRNA gene spacer (ITS) and sequencing

The PCR reaction was performed as described previously by Chang et al. (2005) with a few modifications. The total reaction volume was 25 μl and consisted of 1.0 μM of each primer, 1.5 mM of MgCl₂, 1X buffer, 0.2 mM of each DNTP, and 2U of Taq DNA polymerase. The PCR was performed as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation (94°C for 1 min), annealing (57°C for 1 min), and extension (72°C for 1.5 min), with a final extension step at 72°C for 7 min.

PCR products were purified with EXOSAP‐IT^® enzyme USB and were sequenced using the ABI 3500 Genetic Analyzer with 50 cm capillaries and POP7 polymer (Applied Biosystems, Foster City, CA).

Multiplex PCR for gyrB gene

The isolates were submitted to gyrB multiplex PCR as described by Higgins et al. (2010) with few modifications. The total reaction volume was 25 μl consisting of 0.2 μM of each primer, 1.5 mM of MgCl₂, 1X buffer, 0.2 mM of each DNTP, and 1 U of Taq DNA polymerase. The PCR was run as follows: initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation (94°C for 1 min), annealing (56°C for 30 sec), and extension (72°C for 1 min), with a final extension at 72°C for 10 min. PCR products were analyzed on agarose 1.5% w⁄ v gels, stained with ethidium bromide, and visualized under UV.

Minimal Inhibitory Concentration (MIC) was performed by broth microdilution for Imipenem, Polymyxin B, and Tigeciycline according to criteria of the Clinical and Laboratory Standards Institute (CLSI). Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 were used as quality control.

Results and Discussion

The multiplex PCR for the gyrB gene, identified the majority (106/117–90.6%) of isolates as A. baumannii. 6 (5.1%) and 4 (3.4%) isolates were identified as A. nosocomialis and A. pittii, respectively. Only one isolate did not present a product of the PCR for the gyrB gene; this isolate was identified as Acinetobacter genospecie 10 by sequencing of ITS (Table 1).

Table 1.

Comparison of 16S‐23S ITS and gyrB Methods to Identify Acinetobacter Calcoaceticus‐baumannii Complex Species

Isolate number	ITS	gyrB	Clinical specimen
1nb	A. pittii	A. pittii	Tracheal aspirate
2nb	A. nosocomialis	A. nosocomialis	Blood culture
3nb	A. pittii	A. pittii	Sputum catheter
4nb	A. pittii	A. pittii	Urine
5nb	A. genomic specie 10	Non‐identified a	Sputum catheter
6nb	A. nosocomialis	A. nosocomialis	Tracheal aspirate
7nb	A. nosocomialis	A. nosocomialis	Tracheal aspirate
8nb	A. nosocomialis	A. nosocomialis	Blood culture
9nb	A. pittii	A. pittii	Tracheal aspirate
10nb	A. nosocomialis	A. nosocomialis	Tracheal aspirate
11nb	A. nosocomialis	A. nosocomialis	Tracheal aspirate

^aThere is not product amplified on PCR to gyrB.

There was 100% of concordance of the gyrB PCR with sequencing of the ITS. This technique proved to be able to identify all species of ABC since, only one isolate which was not identified in the multiplex PCR for gyrB gene proved to be A. genospecie 10 when analyzed by sequencing ITS, a specie that does not belong to the ABC complex. Although the sequencing of the ITS could be considered the gold standard to identify species of ABC, the multiplex PCR of the gyrB is a more feasible method to be used in the routine laboratory as a rapid alternative to identification at specie level.

Other methods based on DNA analysis such as hybridization, the 16S rDNA and whole genome sequencing require at least two steps of accomplishment becoming more laborious and expensive to be implemented in the routine laboratory. Moreover, requires sophisticated technologies and equipment that, usually, are restricted to reference laboratories 4, 14. According to our experience, the multiplex PCR for the gyrB could be performed in nearly 3 hr and requires less sophisticated equipment and analysis of the results.

As shown in the literature, the A. baumannii is involved in serious and difficult to treat infections due to high rates of resistance, particularly to carbapenems 16, 17. For other species of ABC complex, non‐A. baumannii, there are still few reports of susceptibility profile and clinical impact 8, 9, 10. In this study, the non‐A. baumannii isolates were recovered from respiratory tract (8/11; 72.7%), blood (2/11; 18.2%), urine (1/11; 9.1%) and 4 of these (36.4%) were resistant to imipenem. In addition to causing infections, ABC may be colonizing of patients with some comorbidity or who are hospitalized and receiving antibiotics. This fact confirmed non‐A. baumannii species may be associated with severe infections and present phenotypes of multidrug resistant as A. baumannii 18, 19 highlighting the importance of correct identification at the species level for a better understanding of the clinical and epidemiological aspects of these infections.

Conflict of Interest

The authors declare have not conflict of interests.