Identification of Acinetobacter Species and Genotyping of Acinetobacte
http://www.100md.com
《微生物临床杂志》
Ibis Biosciences, Isis Pharmaceuticals, Inc., 1891 Rutherford Road, Carlsbad, California 92008
Walter Reed Army Institute for Research, Silver Spring, Maryland 20910
Walter Reed Army Medical Center, Washington, D.C. 20307
Landstuhl Regional Medical Center, Landstuhl, Germany
Aberdeen Proving Ground, Aberdeen, Maryland
Unite Biodiversite des Bacteries Pathogenes Emergentes (U389 INSERM), Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France
ABSTRACT
Members of the genus Acinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotype Acinetobacter isolates to determine epidemiology and clonality during infectious outbreaks. Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) is a method that uses the amplicon base compositions to genotype bacterial species. In order to identify regions of the Acinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE, adk, efp, mutY, fumC, and ppa) from 267 isolates of Acinetobacter. Isolates were collected from infected and colonized soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq, from previously characterized outbreaks in European hospitals, and from culture collections. Most of the isolates from the Iraqi conflict were Acinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterized A. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing different Acinetobacter species, including 8 representatives of Acinetobacter genomospecies 13TU and 13 representatives of Acinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of the trpE, adk, mutY, fumC, and ppa genes distinguished 47 of the 48 A. baumannii genotypes identified by sequencing and identified at the species level at least 18 Acinetobacter species. Results obtained with our genotyping method were essentially in agreement with those obtained by pulse-field gel electrophoresis analysis. The PCR/ESI-MS genotyping method required 4 h of analysis time to first answer with additional samples subsequently analyzed every 10 min. This rapid analysis allows tracking of transmission for the implementation of appropriate infection control measures on a time scale previously not achievable.
INTRODUCTION
Acinetobacter spp. are aerobic gram-negative organisms widely distributed in the soil and water of natural environments (4) and are also important nosocomial pathogens. Acinetobacter outbreaks involving multidrug-resistant strains have occurred worldwide (5, 6, 13, 14, 28, 36, 39, 46, 47). In hospitalized patients, Acinetobacter baumannii frequently colonizes the skin and upper respiratory tract and has been isolated from human sputum, blood, urine, and feces (1, 2, 30). A. baumannii also persists for long periods on hospital environmental surfaces and has been isolated from various locations, including air, tap water faucets, bedside urinals, ventilators, gloves, and angiography catheters (9, 25, 29). Acinetobacter infections have been historically associated with military and civilian personnel injured in combat, possibly due to direct environmental contamination of wounds. For example, during the Vietnam War, A. baumannii was the most common gram-negative bacterium recovered from traumatic injuries to extremities (37).
A recent report described an increasing number of A. baumannii bloodstream infections in patients at military medical facilities that were treating service members injured in the Iraq/Kuwait region during Operation Iraqi Freedom (OIF) and in Afghanistan during Operation Enduring Freedom (32). Over a 2-year period from 2002 to 2004, military health officials identified 102 patients with blood cultures that grew A. baumannii. Most of the infections were reported from the Landstuhl Regional Medical Center (LRMC) in Germany and the Walter Reed Army Medical Center (WRAMC) in the United States. In both facilities, the number of patients with A. baumannii bloodstream infections in 2003 and 2004 significantly exceeded those reported in previous years, suggesting nosocomial transmission. This has the potential to become a serious problem in military and veterans' hospitals, where soldiers returning from active duty worldwide are treated in the same environment as other patients.
Understanding the fundamental mechanisms underlying Acinetobacter infections, including the original sources of the infecting organisms, their clonality, and geographical spread, is an important requirement for the development of appropriate infection control measures. Genotyping allows investigation of clonal spread and can be used to identify the source of the original infection. Traditional Acinetobacter strain typing methods include serotyping (38), multilocus enzyme electrophoresis (34), and DNA-based methods, including repetitive extragenic palindromic sequence-based PCR (8, 19), amplified ribosomal DNA restriction analysis (ARDRA), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) (43), and ribotyping (21, 23, 26). However, these methods are not optimal: despite strenuous efforts at standardization, it is difficult to compare results obtained in different laboratories, and the methods are labor intensive and time consuming. Multilocus sequence typing (MLST) is a high-resolution molecular tool for discriminating between closely related bacterial species (40). MLST data are reproducible and portable, facilitating comparison among laboratories worldwide. MLST approaches to strain typing of Acinetobacter baumannii are currently based on sequencing regions of housekeeping genes (3) or the drug efflux gene adeB (20). However, these MLST approaches cannot provide a real-time surveillance capability. Furthermore, Acinetobacter infections can be the result of one of several pathogenic Acinetobacter species, including A. baumannii, Acinetobacter genomospecies 13TU, or Acinetobacter genomospecies 3 (17, 35, 42, 45).
Here, we describe a very rapid (4 h of analysis time to first answer with additional samples analyzed every 10 min) multilocus method for the species-level identification of Acinetobacter spp. and the genotyping of A. baumannii. PCR amplicons from housekeeping genes are analyzed by electrospray ionization mass spectrometry (PCR/ESI-MS), and the base compositions (the numbers of adenines, guanines, cytosines, and thymines) of the amplicons are determined (15, 16, 31). The present study was performed with a collection of 216 Acinetobacter field isolates and 51 diverse reference strains. Results from the PCR/ESI-MS genotyping assay and PFGE analysis were compared.
MATERIALS AND METHODS
Isolates tested. A total of 267 isolates were analyzed by PCR/ESI-MS and sequencing of the six MLST loci. These included a collection of 23 isolates from European hospitals that were previously characterized by ribotyping, PFGE, and AFLP and that are representative of the major clones, I, II, and III (41). We also obtained 27 isolates from the American Type Culture Collection (ATCC) representing several Acinetobacter species. These were previously characterized by ARDRA, RAPD, and AFLP (23). In addition, 216 strains were isolated as part of an ongoing investigation of A. baumannii infections among patients at military medical facilities where U.S. service personnel were treated between 2002 and 2004 (32). One reference strain analyzed in this study, Acinetobacter baylyi ATCC 33405, was represented by its genomic sequence; this strain was formerly known as Acinetobacter sp. strain ADP1 (44). Partial sequences from strain LUH8731 were also used to characterize Acinetobacter genomospecies 13TU isolates (sequence data kindly provided by Lenie Dijkshoorn).
PCR/ESI-MS gene targets and selection of genotyping primers. The Acinetobacter genes used for PCR/ESI-MS analysis were the same as those used in the Moraxella MLST project (http://web.mpiib-berlin.mpg.de/mlst/dbs/Mcatarrhalis). The genes analyzed were those encoding anthranilate synthase component I (trpE), adenylate kinase (adk), translation elongation factor P (efp), A/G-specific DNA glycosylase (mutY), fumarase (fumC), and inorganic pyrophosphatase (ppa). We created an alignment of all of the sequences for each of these genes from the 267 isolates and concatenated them (total of 1,679 positions, excluding primer pair sequences). These combined sequences were grouped into 85 sequence types, wherein a single nucleotide difference in 1 of the total of 1,679 positions resulted in a new sequence type. Representatives of each sequence type were then analyzed for the design of PCR/ESI-MS genotyping primers, based on criteria previously described (15, 16).
Genome preparation, sequencing, and PCR. Genomic material from cultured samples was prepared with the DNeasy Tissue Kit (QIAGEN, Valencia, CA) according to the manufacturer's protocols. All PCRs were assembled in 40-μl reaction mixtures in the 96-well microtiter plate format by using a Packard MPII liquid handling robotic platform and MJ Dyad thermocyclers (MJ Research, Waltham, MA). Sequencing of PCR-generated amplicons was performed by QIAGEN.
Sequencing of the Acinetobacter trpE, adk, efp, mutY, fumC, and ppa loci was performed with degenerate PCR sequencing primers (Table 1, first-generation sequencing primer pairs) selected for complementarity to the previously sequenced Acinetobacter baylyi strain ATCC 33305. These primers were synthesized with the M13 sequencing primer targets at their 5' ends to facilitate direct sequencing of the PCR amplicons. The sequencing primers were used to generate amplicons from a diverse subset of Acinetobacter strains: ATCC strains 27227, 17976, 17908, 17904, 15309, and 9955 and OIF strains 197, 148, 147, 146, and 140. Due to the genetic diversity of these strains it was necessary to employ a range of PCR conditions to generate amplicons from all strains. The PCR mix consisted of 4 units of AmpliTaq Gold, 1x buffer II (Applied Biosystems, Foster City, CA), 2 to 3 mM MgCl2, 0.4 M betaine, 800 μM deoxynucleoside triphosphate (dNTP) mix, and 250 nM (each) primer. The following PCR conditions were used to generate the amplicons for sequencing: 96°C for 10 min, followed by eight cycles of 96°C for 15 seconds, 52 to 58°C for 20 to 90 seconds, and 72°C for 20 seconds. The PCR was then continued for 37 additional cycles of 96°C for 15 seconds, 63°C for 10 seconds, and 72°C for 20 seconds.
Based on the Acinetobacter sequences generated with the first generation sequencing primers, a set of second-generation sequencing primers (Table 1) was designed and used to sequence the PCR/ESI-MS target loci of all isolates. For these reactions, the PCR mix consisted of 4 units of AmpliTaq Gold, 1x buffer II, 2 mM MgCl2, 0.4 M betaine, 800 μM dNTP mix, and 250 nM (each) primer. The following PCR conditions were used to generate the amplicons for sequencing: 96°C for 10 min, followed by eight cycles of 95°C for 15 seconds, 52°C for 90 seconds, and 72°C for 40 seconds, with the 52°C annealing temperature increasing by 0.6°C each cycle. The PCR was then continued for 37 additional cycles of 96°C for 15 seconds, 58°C for 20 seconds, and 72°C for 40 seconds.
The primers used for PCR/ESI-MS analysis are shown in Table 1. The PCR mix consisted of 4 units of AmpliTaq Gold, 1x buffer II, 1.5 mM MgCl2, 0.4 M betaine, 800 μM dNTP mix, and 250 nM (each) primer. The following PCR conditions were used to amplify the sequences used for PCR/ESI-MS analysis: 95°C for 10 min followed by eight cycles of 95°C for 30 seconds, 48°C for 30 seconds, and 72°C for 30 seconds, with the 48°C annealing temperature increasing by 0.9°C each cycle. The PCR was then continued for 37 additional cycles of 95°C for 15 seconds, 56°C for 20 seconds, and 72°C for 20 seconds.
Phylogenetic analysis. Neighbor-joining trees were constructed with the dnadist and neighbor programs of the Phylip package (http://evolution.genetics.washington.edu/phylip.html). The efp tree was assembled with sequence from the efp gene from all Acinetobacter species. For the Acinetobacter species with multiple isolates in our collection, sequences were selected to represent the allele diversity present in the efp gene (A. baumannii OIF007, OIF064, OIF139, OIF143, OIF319, OIF321, OIF327, and ATCC 17904; Acinetobacter genomospecies 13TU OIF145 and ATCC 17903; and Acinetobacter genomospecies 3 OIF061, OIF131, OIF308, OIF323, OIF325, and ATCC 19004).
Representatives of the 48 A. baumannii sequence types were used for the A. baumannii tree. Both trees were bootstrapped with the seqboot and consense programs of the Phylip package and were analyzed with treeview (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Final editions were created with Adobe Illustrator.
MS and base composition analysis. Following amplification, 15-μl aliquots of each PCR mix were desalted and with a weak anion-exchange protocol as described elsewhere (22). Accurate mass (±1 ppm) and high-resolution (M/dM > 100,000 full-width half-maximum resolution) mass spectra were acquired for each sample by the PCR/ESI-MS protocols described previously (16). For each sample, approximately 1.5 μl of analyte solution was consumed during the 74-second spectral acquisition. Raw mass spectra were postcalibrated with an internal mass standard and deconvolved to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides (27). Quantitative results were obtained by comparing the peak heights with an internal PCR calibration standard present in every PCR well at 100 molecules (16).
PFGE. The PFGE protocol, described at http://www.cdc.gov/pulsenet/protocols.htm, was used with modifications for Acinetobacter. Bacterial DNA was prepared with a Salmonella standard and ApaI and XbaI restriction enzymes for Acinetobacter and Salmonella, respectively. Restriction fragments were separated with a contour-clamped homogenous electric field electrophoresis instrument (CHEF Mapper; Bio-Rad Laboratories). PFGE conditions were 6.0 V/cm gradient, 120° angle, 14°C, and 7- to 20-second pulse times for 18.5 h. Gel images were entered into the Bionumerics software (Applied Maths, Kortrijk, Belgium) for band analysis with the Dice coefficient. The unweighted pair group method with averages, with a position tolerance for comparison of 1.5%, was used to produce the dendrogram. Strain sameness was defined as greater than or equal to 90% relatedness.
RESULTS
Diversity of PCR/ESI-MS target genes for Acinetobacter. To identify a set of genes suitable for creation of an PCR/ESI-MS database for Acinetobacter, we selected the set of housekeeping genes used in the Moraxella catarrhalis MLST database. Moraxella is the organism closest to Acinetobacter for which an MLST database exists (both Acinetobacter and Moraxella are members of the family Moraxellaceae). Using primer pairs derived from the completely sequenced Acinetobacter baylyi strain, homologous regions of the housekeeping genes trpE, adk, efp, mutY, fumC, and ppa were sequenced from 267 Acinetobacter isolates collected from outbreaks in European hospitals, from samples associated with the military conflict in Iraq, and from reference strains in the ATCC culture collection. This set of diverse isolates contains several different Acinetobacter species, including representatives of the major clones, I, II, and III, obtained from the European hospital collection (12, 41) Target gene sequences from these isolates showed a considerable range of allelic variation (Fig. 1), virtually all (>98%) due to silent mutations. The efp gene was the most conserved and showed very little variation among A. baumannii isolates, suggesting that the efp gene is under more stringent evolutionary constraints that the other genes analyzed. As a result, the efp gene was the least useful of the six target genes for parsing A. baumannii isolates into different subgroups. Only eight alleles were indicated by the efp gene (Fig. 1), and the sequences differed from the consensus sequence by single point mutations. However, the efp gene had significant allelic variation among different species of Acinetobacter and was the most useful among the six target genes for characterizing Acinetobacter isolates at the species level. The other five genes, mutY (25 alleles), fumC (25 alleles), trpE (21 alleles), adk (13 alleles), and ppa (14 alleles), had significant sequence diversity at the subspecies level for A. baumannii (Fig. 2) and were used to design nine primer pairs for PCR/ESI-MS analysis (one each for trpE, adk, efp, and ppa, two for mutY, and three for fumC).
Analysis of isolates by PCR/ESI-MS. The entire collection of 267 isolates was analyzed by PCR/ESI-MS; as expected, the base compositions determined experimentally were in agreement with the sequence-derived base compositions (summarized in Fig. 2). The relatedness of the isolates was assessed by using sequences from the efp gene, the gene in the set for which PCR amplicons for sequencing could be obtained for all isolates tested (including the non-baumannii Acinetobacter species) (Fig. 3, top). The uniqueness of the different Acinetobacter species is especially visible in base composition space of amplicons generated with the efp PCR/ESI-MS primer pair (Fig. 3, bottom). The majority of OIF samples (Fig. 2) fell into three tight clusters designated A. baumannii (189 isolates), Acinetobacter genomospecies 13TU (8 isolates), and Acinetobacter genomospecies strain 3 (13 isolates).
A. baumannii genotyping. The 221 A. baumannii isolates (189 OIF strains, 23 European strains, and 9 ATCC strains) are depicted in Fig. 3 as a tight wedge and are shown on an expanded scale in Fig. 4. This group, although numerically abundant in this sample set, was tightly clustered on the efp phylogenetic tree. These A. baumannii isolates cluster into 48 different sequencing genotypes that are distinguished by 116 mutations, significantly fewer than the 531 positions that exhibit variation within all of the Acinetobacter isolates evaluated. Twenty-one of the 48 A. baumannii sequence types were represented by two or more isolates of identical genotype. Notably, of the 48 types identified by sequencing, 47 types were also distinguishable by PCR/ESI-MS using the set of nine PCR primer pairs. The one strain type identified only by sequence was a single-isolate variant of type ST12 (LUH6011). This reference strain was distinguished within the ppa locus, by a C-to-T and a T-to-C mutation, from the nine other ST12 reference strains; these simultaneous mutations did not change the amplicon mass and were therefore not distinguished by PCR/ESI-MS.
Clusters of related types that share four or five alleles are readily apparent. In particular, the first cluster contained the most-represented type (ST11, 51 isolates) and mutY variants in types ST1, ST10, ST12, and ST47. A second cluster consisted of types ST15, ST16, ST45, and ST46. Similarly, types ST14 and ST8 differed only by a single mutation in the fumC gene. These clusters are highlighted in red and orange in Fig. 1, 2, and 4.
The A. baumannii group included nine reference strains from the ATCC and 23 well-characterized European A. baumannii strains, with representatives of European hospital clones I and II and the recently identified pan-European multidrug-resistant clone III (represented in blue in Fig. 4) (12, 41). Twelve of the 23 European strains were identical at all six loci studied with one or more OIF isolates, providing a direct comparison between ribogroup and PCR/ESI-MS typing (Fig. 2). In particular, type ST15 contained seven OIF isolates and five clone I European isolates. Cluster ST14 contained 22 OIF isolates and 6 clone III European isolates. No OIF isolate was found to be identical to clone II European isolates, found exclusively in ST12. However, 51 OIF isolates differed from clone II by only four mutations at the mutY locus. Overall, these data strongly suggest that the 111 OIF isolates belonging to the ST15/16/45/46, ST1/10/11/12, and ST8/14 clusters are phylogenetically associated with European hospital clones I, II, and III, respectively.
Characterization of other Acinetobacter species. Two groups of OIF isolates were distinct from the A. baumannii isolates and matched the reference strains for Acinetobacter genomospecies 13TU and Acinetobacter genomospecies 3 (Fig. 3). The Acinetobacter genomospecies 13TU group contained eight OIF isolates (seven patient samples from Walter Reed Army Medical Center and Landstuhl Regional Medical Center and one environmental sample) and reference strain ATCC 17903 and was comprised of five unique genotypes, A1 to A5. The Acinetobacter genomospecies 3 group contained 13 OIF isolates (nine environmental and four patient samples) and reference strain ATCC 19004 and was comprised of genotypes B1 to B10. The assignment of these isolates to different species was firmly established due to the uniqueness of the base composition signatures (in Fig. 2, note how the olive and bright green base compositions are restricted to these species).
Six OIF isolates (OIF129, OIF136, OIF199, OIF301, OIF304, and OIF306) were genotypically interspersed with the diverse non-baumannii Acinetobacter reference strains (Fig. 3). A. baumannii is the predominant species of Acinetobacter that causes human disease, but many other species of this genus have also been shown to infect humans (14, 24). Isolate OIF129 was assigned to the species A. ursingii, based on the base composition and sequence homology found in both efp (variation at a single position between OIF129 and the reference strain BAA-617) and trpE (seven variations). Isolates OIF136 and OIF306 were similarly assigned to the species A. haemolyticus and Acinetobacter genomospecies 10, respectively.
Relationship between PCR/ESI-MS and PFGE genotyping. A comparison of the classifications obtained by PCR/ESI-MS typing and PFGE is shown in Fig. 5. Dual classification was available for 200 isolates, mapped to 57 pies representing the number of sequence types; pies were then sliced to reflect the classification of these isolates into the 66 observed PFGE types. Fifty-two sequence types and PFGE types were clearly correlated and provided a consistent classification for 164 isolates (82% of the total evaluated [green in Fig. 5]). Twenty-one isolates were further discriminated by PFGE (Fig. 5, yellow). In particular, isolates belonging to ST14 were almost equally split into two PFGE types regardless of origin, suggesting that the PCR/ESI-MS genotyping method could be further improved for higher resolution. Conversely, five isolates were resolved by PCR/ESI-MS and not by PFGE (Fig. 5, blue pies) indicating that strain resolution by PFGE is also not exhaustive. For example, PFGE type 23 was composed of the closely related types ST28 and ST38. Isolates OIF123 and OIF129, characterized by PCR/ESI-MS as non-baumannii Acinetobacter species, shared PFGE types with A. baumannii isolates belonging to ST11 and ST30, respectively. PFGE and the PCR/ESI-MS genotyping methods were not in agreement for the 10 remaining isolates (Fig. 5, red).
DISCUSSION
Acinetobacter baumannii is a pathogen that has been associated with hospital outbreaks worldwide (6, 7, 9, 11, 18, 28, 33). This organism is particularly problematic due to the large number of multidrug-resistant strains, including the pan-European clone types I, II, and III, that have become endemic in hospital settings (12, 41). Recently, infections by Acinetobacter baumannii have resulted in critical complications for troops injured in Iraq and Afghanistan (47). The injured were treated in a variety of locations in the field and in European hospitals. As part of a public health investigation to identify the possible source(s) of this outbreak, to determine the likely mode(s) of transmission, and to assist in generating and implementing infection control countermeasures, we set out to develop a rapid Acinetobacter baumannii strain typing assay. The assay we developed is similar to traditional MLST strain typing, but analysis of PCR amplicons is performed with ESI-MS rather than standard DNA sequencing. The base composition of the PCR amplicons is determined from the masses of the PCR amplicons and provides enough information to type the strains. Previously we developed a similar assay for the rapid strain typing of respiratory pathogens (15, 16).
To develop the A. baumannii typing assay, we assembled a collection of 267 diverse isolates. Included in this collection were 216 isolates from U.S. and Iraqi patients involved in an outbreak in U.S. military hospitals associated with OIF, 23 isolates containing representatives of the endemic pan-European clones types I, II, and III, and a diverse collection of commercially available clones representing Acinetobacter baumannii and other Acinetobacter species.
Analysis of the collection of 216 Acinetobacter isolates from U.S. and Iraqi patients treated in U.S. military hospitals identified 31 (14.4%) A. baumannii isolates that were indistinguishable from the European reference strains that are plaguing European hospitals. Additionally, 80 OIF isolates (37%) differed from the endemic European reference strains by minor variations at a single locus, suggesting a relationship with the European endemic strains. The remaining 105 isolates represented more diverse strains.
Twenty-seven OIF Acinetobacter isolates (12.5%) were found to represent at least seven additional Acinetobacter species. Two of these were represented by multiple isolates and displayed intraspecies allelic variation; we were able to identify these two species as Acinetobacter genomospecies 13TU and Acinetobacter genomospecies 3 through comparison with relevant reference strains. Interestingly, these results mirror a recent study that identified the same two species amid infections by A. baumannii in hospital settings (10).
Three OIF isolates did not match any of our reference species and may represent novel Acinetobacter species. Due to sequence variation of the non-baumannii Acinetobacter isolates, we were not able to generate PCR/ESI-MS or sequencing PCR amplicons for these isolates from all primer pairs. Based upon the efp sequence and PCR/ESI-MS analyses from the other loci, we determined that these isolates were not Acinetobacter baumannii and did not match any of the 18 representatives of diverse Acinetobacter species tested. The unexpected diversity of these isolates highlights the potential of the PCR/ESI-MS approach to detect and strain type de novo infections from other Acinetobacter species.
Sequence analysis of the entire strain collection, including the European reference strains and the commercially available strains, identified 48 unique A. baumannii strain types. Interestingly, only one of the nine A. baumannii reference strains from the ATCC collection (ATCC 9955, ST9) was represented in the OIF isolates, suggesting that the bulk of the OIF sequences are distinct from the strains archived at the ATCC.
Base composition, though not as information rich as sequence data, has nearly the same resolving power for Acinetobacter baumannii strain typing as sequencing. By selecting primer pairs targeting nine information-rich regions of six loci, we were able to resolve by PCR/ESI-MS 47 of the 48 Acinetobacter baumannii strain types identified by sequencing 1,679 nucleotides from the six loci. The only case in which PCR/ESI-MS analysis did not resolve the strain type involved the two mutations (C to T and T to C) that distinguish isolate LUH6011 from the other clone III isolates: these mutations are mutually compensating, resulting in no net change in the PCR amplicon mass. Additional primers specifically targeting this isolate would bring no value to the resolution of other types, as the use of these nine primer pairs had already maximized the average number of mutations between all characterized types.
Our sequence analysis showed that the efp gene was not useful for discrimination of Acinetobacter baumannii isolates (Fig. 1), the initial goal of this study. However, the efp gene was particularly useful for discriminating Acinetobacter strains at the species level. The incorporation of the efp primer pair expanded the discrimination power of the PCR/ESI-MS Acinetobacter baumannii MLST assay to include the identification and typing of other Acinetobacter species.
In summary, we have used an A. baumannii MLST-like strain typing assay based on PCR/ESI-MS of nine PCR amplicons to identify 47 A. baumannii strain types. Fifteen additional types represented the related Acinetobacter genomospecies 13TU and Acinetobacter genomospecies 3. Using a portion of the efp locus, we distinguished at least 16 additional Acinetobacter species. The PCR/ESI-MS strain typing method is both rapid and low cost and can be performed in an automated modality, enabling the rapid surveillance of Acinetobacter spp. in a variety of settings. Use of this A. baumannii typing assay identified a genetic relationship between the endemic European isolates and many of the isolates found in patients and in military hospitals treating casualties from OIF. Further analysis of the OIF isolates and their locations of collection should provide better understanding of the origins of these infections and will ultimately improve infection control and prevention measures.
ACKNOWLEDGMENTS
We acknowledge DARPA and the CDC for financial support.
We thank Kyle Petersen for contributing Acinetobacter isolates from patients under his care on the U.S. Navy ship (hospital) Comfort while deployed in the Persian Gulf. We also acknowledge Amy Summers, of the Walter Reed Army Medical Center, for technical support in culturing and reisolation of strains used in this study and Lenie Dijkshoorn for providing reference sequences of Acinetobacter sp. strain 13TU.
FOOTNOTES
Corresponding author. Mailing address: Ibis Biosciences, Isis Pharmaceuticals, Inc., 1891 Rutherford Road, Carlsbad, CA 92008. Phone: (760) 603-3892. Fax: (760) 603-4653. E-mail: meshoo@isisph.com.
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Walter Reed Army Institute for Research, Silver Spring, Maryland 20910
Walter Reed Army Medical Center, Washington, D.C. 20307
Landstuhl Regional Medical Center, Landstuhl, Germany
Aberdeen Proving Ground, Aberdeen, Maryland
Unite Biodiversite des Bacteries Pathogenes Emergentes (U389 INSERM), Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris Cedex 15, France
ABSTRACT
Members of the genus Acinetobacter are ubiquitous in soil and water and are an important cause of nosocomial infections. A rapid method is needed to genotype Acinetobacter isolates to determine epidemiology and clonality during infectious outbreaks. Multilocus PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) is a method that uses the amplicon base compositions to genotype bacterial species. In order to identify regions of the Acinetobacter genome useful for this method, we sequenced regions of six housekeeping genes (trpE, adk, efp, mutY, fumC, and ppa) from 267 isolates of Acinetobacter. Isolates were collected from infected and colonized soldiers and civilians involved in an outbreak in the military health care system associated with the conflict in Iraq, from previously characterized outbreaks in European hospitals, and from culture collections. Most of the isolates from the Iraqi conflict were Acinetobacter baumannii (189 of 216 isolates). Among these, 111 isolates had genotypes identical or very similar to those associated with well-characterized A. baumannii isolates from European hospitals. Twenty-seven isolates from the conflict were found to have genotypes representing different Acinetobacter species, including 8 representatives of Acinetobacter genomospecies 13TU and 13 representatives of Acinetobacter genomospecies 3. Analysis by the PCR/ESI-MS method using nine primer pairs targeting the most information-rich regions of the trpE, adk, mutY, fumC, and ppa genes distinguished 47 of the 48 A. baumannii genotypes identified by sequencing and identified at the species level at least 18 Acinetobacter species. Results obtained with our genotyping method were essentially in agreement with those obtained by pulse-field gel electrophoresis analysis. The PCR/ESI-MS genotyping method required 4 h of analysis time to first answer with additional samples subsequently analyzed every 10 min. This rapid analysis allows tracking of transmission for the implementation of appropriate infection control measures on a time scale previously not achievable.
INTRODUCTION
Acinetobacter spp. are aerobic gram-negative organisms widely distributed in the soil and water of natural environments (4) and are also important nosocomial pathogens. Acinetobacter outbreaks involving multidrug-resistant strains have occurred worldwide (5, 6, 13, 14, 28, 36, 39, 46, 47). In hospitalized patients, Acinetobacter baumannii frequently colonizes the skin and upper respiratory tract and has been isolated from human sputum, blood, urine, and feces (1, 2, 30). A. baumannii also persists for long periods on hospital environmental surfaces and has been isolated from various locations, including air, tap water faucets, bedside urinals, ventilators, gloves, and angiography catheters (9, 25, 29). Acinetobacter infections have been historically associated with military and civilian personnel injured in combat, possibly due to direct environmental contamination of wounds. For example, during the Vietnam War, A. baumannii was the most common gram-negative bacterium recovered from traumatic injuries to extremities (37).
A recent report described an increasing number of A. baumannii bloodstream infections in patients at military medical facilities that were treating service members injured in the Iraq/Kuwait region during Operation Iraqi Freedom (OIF) and in Afghanistan during Operation Enduring Freedom (32). Over a 2-year period from 2002 to 2004, military health officials identified 102 patients with blood cultures that grew A. baumannii. Most of the infections were reported from the Landstuhl Regional Medical Center (LRMC) in Germany and the Walter Reed Army Medical Center (WRAMC) in the United States. In both facilities, the number of patients with A. baumannii bloodstream infections in 2003 and 2004 significantly exceeded those reported in previous years, suggesting nosocomial transmission. This has the potential to become a serious problem in military and veterans' hospitals, where soldiers returning from active duty worldwide are treated in the same environment as other patients.
Understanding the fundamental mechanisms underlying Acinetobacter infections, including the original sources of the infecting organisms, their clonality, and geographical spread, is an important requirement for the development of appropriate infection control measures. Genotyping allows investigation of clonal spread and can be used to identify the source of the original infection. Traditional Acinetobacter strain typing methods include serotyping (38), multilocus enzyme electrophoresis (34), and DNA-based methods, including repetitive extragenic palindromic sequence-based PCR (8, 19), amplified ribosomal DNA restriction analysis (ARDRA), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) (43), and ribotyping (21, 23, 26). However, these methods are not optimal: despite strenuous efforts at standardization, it is difficult to compare results obtained in different laboratories, and the methods are labor intensive and time consuming. Multilocus sequence typing (MLST) is a high-resolution molecular tool for discriminating between closely related bacterial species (40). MLST data are reproducible and portable, facilitating comparison among laboratories worldwide. MLST approaches to strain typing of Acinetobacter baumannii are currently based on sequencing regions of housekeeping genes (3) or the drug efflux gene adeB (20). However, these MLST approaches cannot provide a real-time surveillance capability. Furthermore, Acinetobacter infections can be the result of one of several pathogenic Acinetobacter species, including A. baumannii, Acinetobacter genomospecies 13TU, or Acinetobacter genomospecies 3 (17, 35, 42, 45).
Here, we describe a very rapid (4 h of analysis time to first answer with additional samples analyzed every 10 min) multilocus method for the species-level identification of Acinetobacter spp. and the genotyping of A. baumannii. PCR amplicons from housekeeping genes are analyzed by electrospray ionization mass spectrometry (PCR/ESI-MS), and the base compositions (the numbers of adenines, guanines, cytosines, and thymines) of the amplicons are determined (15, 16, 31). The present study was performed with a collection of 216 Acinetobacter field isolates and 51 diverse reference strains. Results from the PCR/ESI-MS genotyping assay and PFGE analysis were compared.
MATERIALS AND METHODS
Isolates tested. A total of 267 isolates were analyzed by PCR/ESI-MS and sequencing of the six MLST loci. These included a collection of 23 isolates from European hospitals that were previously characterized by ribotyping, PFGE, and AFLP and that are representative of the major clones, I, II, and III (41). We also obtained 27 isolates from the American Type Culture Collection (ATCC) representing several Acinetobacter species. These were previously characterized by ARDRA, RAPD, and AFLP (23). In addition, 216 strains were isolated as part of an ongoing investigation of A. baumannii infections among patients at military medical facilities where U.S. service personnel were treated between 2002 and 2004 (32). One reference strain analyzed in this study, Acinetobacter baylyi ATCC 33405, was represented by its genomic sequence; this strain was formerly known as Acinetobacter sp. strain ADP1 (44). Partial sequences from strain LUH8731 were also used to characterize Acinetobacter genomospecies 13TU isolates (sequence data kindly provided by Lenie Dijkshoorn).
PCR/ESI-MS gene targets and selection of genotyping primers. The Acinetobacter genes used for PCR/ESI-MS analysis were the same as those used in the Moraxella MLST project (http://web.mpiib-berlin.mpg.de/mlst/dbs/Mcatarrhalis). The genes analyzed were those encoding anthranilate synthase component I (trpE), adenylate kinase (adk), translation elongation factor P (efp), A/G-specific DNA glycosylase (mutY), fumarase (fumC), and inorganic pyrophosphatase (ppa). We created an alignment of all of the sequences for each of these genes from the 267 isolates and concatenated them (total of 1,679 positions, excluding primer pair sequences). These combined sequences were grouped into 85 sequence types, wherein a single nucleotide difference in 1 of the total of 1,679 positions resulted in a new sequence type. Representatives of each sequence type were then analyzed for the design of PCR/ESI-MS genotyping primers, based on criteria previously described (15, 16).
Genome preparation, sequencing, and PCR. Genomic material from cultured samples was prepared with the DNeasy Tissue Kit (QIAGEN, Valencia, CA) according to the manufacturer's protocols. All PCRs were assembled in 40-μl reaction mixtures in the 96-well microtiter plate format by using a Packard MPII liquid handling robotic platform and MJ Dyad thermocyclers (MJ Research, Waltham, MA). Sequencing of PCR-generated amplicons was performed by QIAGEN.
Sequencing of the Acinetobacter trpE, adk, efp, mutY, fumC, and ppa loci was performed with degenerate PCR sequencing primers (Table 1, first-generation sequencing primer pairs) selected for complementarity to the previously sequenced Acinetobacter baylyi strain ATCC 33305. These primers were synthesized with the M13 sequencing primer targets at their 5' ends to facilitate direct sequencing of the PCR amplicons. The sequencing primers were used to generate amplicons from a diverse subset of Acinetobacter strains: ATCC strains 27227, 17976, 17908, 17904, 15309, and 9955 and OIF strains 197, 148, 147, 146, and 140. Due to the genetic diversity of these strains it was necessary to employ a range of PCR conditions to generate amplicons from all strains. The PCR mix consisted of 4 units of AmpliTaq Gold, 1x buffer II (Applied Biosystems, Foster City, CA), 2 to 3 mM MgCl2, 0.4 M betaine, 800 μM deoxynucleoside triphosphate (dNTP) mix, and 250 nM (each) primer. The following PCR conditions were used to generate the amplicons for sequencing: 96°C for 10 min, followed by eight cycles of 96°C for 15 seconds, 52 to 58°C for 20 to 90 seconds, and 72°C for 20 seconds. The PCR was then continued for 37 additional cycles of 96°C for 15 seconds, 63°C for 10 seconds, and 72°C for 20 seconds.
Based on the Acinetobacter sequences generated with the first generation sequencing primers, a set of second-generation sequencing primers (Table 1) was designed and used to sequence the PCR/ESI-MS target loci of all isolates. For these reactions, the PCR mix consisted of 4 units of AmpliTaq Gold, 1x buffer II, 2 mM MgCl2, 0.4 M betaine, 800 μM dNTP mix, and 250 nM (each) primer. The following PCR conditions were used to generate the amplicons for sequencing: 96°C for 10 min, followed by eight cycles of 95°C for 15 seconds, 52°C for 90 seconds, and 72°C for 40 seconds, with the 52°C annealing temperature increasing by 0.6°C each cycle. The PCR was then continued for 37 additional cycles of 96°C for 15 seconds, 58°C for 20 seconds, and 72°C for 40 seconds.
The primers used for PCR/ESI-MS analysis are shown in Table 1. The PCR mix consisted of 4 units of AmpliTaq Gold, 1x buffer II, 1.5 mM MgCl2, 0.4 M betaine, 800 μM dNTP mix, and 250 nM (each) primer. The following PCR conditions were used to amplify the sequences used for PCR/ESI-MS analysis: 95°C for 10 min followed by eight cycles of 95°C for 30 seconds, 48°C for 30 seconds, and 72°C for 30 seconds, with the 48°C annealing temperature increasing by 0.9°C each cycle. The PCR was then continued for 37 additional cycles of 95°C for 15 seconds, 56°C for 20 seconds, and 72°C for 20 seconds.
Phylogenetic analysis. Neighbor-joining trees were constructed with the dnadist and neighbor programs of the Phylip package (http://evolution.genetics.washington.edu/phylip.html). The efp tree was assembled with sequence from the efp gene from all Acinetobacter species. For the Acinetobacter species with multiple isolates in our collection, sequences were selected to represent the allele diversity present in the efp gene (A. baumannii OIF007, OIF064, OIF139, OIF143, OIF319, OIF321, OIF327, and ATCC 17904; Acinetobacter genomospecies 13TU OIF145 and ATCC 17903; and Acinetobacter genomospecies 3 OIF061, OIF131, OIF308, OIF323, OIF325, and ATCC 19004).
Representatives of the 48 A. baumannii sequence types were used for the A. baumannii tree. Both trees were bootstrapped with the seqboot and consense programs of the Phylip package and were analyzed with treeview (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Final editions were created with Adobe Illustrator.
MS and base composition analysis. Following amplification, 15-μl aliquots of each PCR mix were desalted and with a weak anion-exchange protocol as described elsewhere (22). Accurate mass (±1 ppm) and high-resolution (M/dM > 100,000 full-width half-maximum resolution) mass spectra were acquired for each sample by the PCR/ESI-MS protocols described previously (16). For each sample, approximately 1.5 μl of analyte solution was consumed during the 74-second spectral acquisition. Raw mass spectra were postcalibrated with an internal mass standard and deconvolved to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides (27). Quantitative results were obtained by comparing the peak heights with an internal PCR calibration standard present in every PCR well at 100 molecules (16).
PFGE. The PFGE protocol, described at http://www.cdc.gov/pulsenet/protocols.htm, was used with modifications for Acinetobacter. Bacterial DNA was prepared with a Salmonella standard and ApaI and XbaI restriction enzymes for Acinetobacter and Salmonella, respectively. Restriction fragments were separated with a contour-clamped homogenous electric field electrophoresis instrument (CHEF Mapper; Bio-Rad Laboratories). PFGE conditions were 6.0 V/cm gradient, 120° angle, 14°C, and 7- to 20-second pulse times for 18.5 h. Gel images were entered into the Bionumerics software (Applied Maths, Kortrijk, Belgium) for band analysis with the Dice coefficient. The unweighted pair group method with averages, with a position tolerance for comparison of 1.5%, was used to produce the dendrogram. Strain sameness was defined as greater than or equal to 90% relatedness.
RESULTS
Diversity of PCR/ESI-MS target genes for Acinetobacter. To identify a set of genes suitable for creation of an PCR/ESI-MS database for Acinetobacter, we selected the set of housekeeping genes used in the Moraxella catarrhalis MLST database. Moraxella is the organism closest to Acinetobacter for which an MLST database exists (both Acinetobacter and Moraxella are members of the family Moraxellaceae). Using primer pairs derived from the completely sequenced Acinetobacter baylyi strain, homologous regions of the housekeeping genes trpE, adk, efp, mutY, fumC, and ppa were sequenced from 267 Acinetobacter isolates collected from outbreaks in European hospitals, from samples associated with the military conflict in Iraq, and from reference strains in the ATCC culture collection. This set of diverse isolates contains several different Acinetobacter species, including representatives of the major clones, I, II, and III, obtained from the European hospital collection (12, 41) Target gene sequences from these isolates showed a considerable range of allelic variation (Fig. 1), virtually all (>98%) due to silent mutations. The efp gene was the most conserved and showed very little variation among A. baumannii isolates, suggesting that the efp gene is under more stringent evolutionary constraints that the other genes analyzed. As a result, the efp gene was the least useful of the six target genes for parsing A. baumannii isolates into different subgroups. Only eight alleles were indicated by the efp gene (Fig. 1), and the sequences differed from the consensus sequence by single point mutations. However, the efp gene had significant allelic variation among different species of Acinetobacter and was the most useful among the six target genes for characterizing Acinetobacter isolates at the species level. The other five genes, mutY (25 alleles), fumC (25 alleles), trpE (21 alleles), adk (13 alleles), and ppa (14 alleles), had significant sequence diversity at the subspecies level for A. baumannii (Fig. 2) and were used to design nine primer pairs for PCR/ESI-MS analysis (one each for trpE, adk, efp, and ppa, two for mutY, and three for fumC).
Analysis of isolates by PCR/ESI-MS. The entire collection of 267 isolates was analyzed by PCR/ESI-MS; as expected, the base compositions determined experimentally were in agreement with the sequence-derived base compositions (summarized in Fig. 2). The relatedness of the isolates was assessed by using sequences from the efp gene, the gene in the set for which PCR amplicons for sequencing could be obtained for all isolates tested (including the non-baumannii Acinetobacter species) (Fig. 3, top). The uniqueness of the different Acinetobacter species is especially visible in base composition space of amplicons generated with the efp PCR/ESI-MS primer pair (Fig. 3, bottom). The majority of OIF samples (Fig. 2) fell into three tight clusters designated A. baumannii (189 isolates), Acinetobacter genomospecies 13TU (8 isolates), and Acinetobacter genomospecies strain 3 (13 isolates).
A. baumannii genotyping. The 221 A. baumannii isolates (189 OIF strains, 23 European strains, and 9 ATCC strains) are depicted in Fig. 3 as a tight wedge and are shown on an expanded scale in Fig. 4. This group, although numerically abundant in this sample set, was tightly clustered on the efp phylogenetic tree. These A. baumannii isolates cluster into 48 different sequencing genotypes that are distinguished by 116 mutations, significantly fewer than the 531 positions that exhibit variation within all of the Acinetobacter isolates evaluated. Twenty-one of the 48 A. baumannii sequence types were represented by two or more isolates of identical genotype. Notably, of the 48 types identified by sequencing, 47 types were also distinguishable by PCR/ESI-MS using the set of nine PCR primer pairs. The one strain type identified only by sequence was a single-isolate variant of type ST12 (LUH6011). This reference strain was distinguished within the ppa locus, by a C-to-T and a T-to-C mutation, from the nine other ST12 reference strains; these simultaneous mutations did not change the amplicon mass and were therefore not distinguished by PCR/ESI-MS.
Clusters of related types that share four or five alleles are readily apparent. In particular, the first cluster contained the most-represented type (ST11, 51 isolates) and mutY variants in types ST1, ST10, ST12, and ST47. A second cluster consisted of types ST15, ST16, ST45, and ST46. Similarly, types ST14 and ST8 differed only by a single mutation in the fumC gene. These clusters are highlighted in red and orange in Fig. 1, 2, and 4.
The A. baumannii group included nine reference strains from the ATCC and 23 well-characterized European A. baumannii strains, with representatives of European hospital clones I and II and the recently identified pan-European multidrug-resistant clone III (represented in blue in Fig. 4) (12, 41). Twelve of the 23 European strains were identical at all six loci studied with one or more OIF isolates, providing a direct comparison between ribogroup and PCR/ESI-MS typing (Fig. 2). In particular, type ST15 contained seven OIF isolates and five clone I European isolates. Cluster ST14 contained 22 OIF isolates and 6 clone III European isolates. No OIF isolate was found to be identical to clone II European isolates, found exclusively in ST12. However, 51 OIF isolates differed from clone II by only four mutations at the mutY locus. Overall, these data strongly suggest that the 111 OIF isolates belonging to the ST15/16/45/46, ST1/10/11/12, and ST8/14 clusters are phylogenetically associated with European hospital clones I, II, and III, respectively.
Characterization of other Acinetobacter species. Two groups of OIF isolates were distinct from the A. baumannii isolates and matched the reference strains for Acinetobacter genomospecies 13TU and Acinetobacter genomospecies 3 (Fig. 3). The Acinetobacter genomospecies 13TU group contained eight OIF isolates (seven patient samples from Walter Reed Army Medical Center and Landstuhl Regional Medical Center and one environmental sample) and reference strain ATCC 17903 and was comprised of five unique genotypes, A1 to A5. The Acinetobacter genomospecies 3 group contained 13 OIF isolates (nine environmental and four patient samples) and reference strain ATCC 19004 and was comprised of genotypes B1 to B10. The assignment of these isolates to different species was firmly established due to the uniqueness of the base composition signatures (in Fig. 2, note how the olive and bright green base compositions are restricted to these species).
Six OIF isolates (OIF129, OIF136, OIF199, OIF301, OIF304, and OIF306) were genotypically interspersed with the diverse non-baumannii Acinetobacter reference strains (Fig. 3). A. baumannii is the predominant species of Acinetobacter that causes human disease, but many other species of this genus have also been shown to infect humans (14, 24). Isolate OIF129 was assigned to the species A. ursingii, based on the base composition and sequence homology found in both efp (variation at a single position between OIF129 and the reference strain BAA-617) and trpE (seven variations). Isolates OIF136 and OIF306 were similarly assigned to the species A. haemolyticus and Acinetobacter genomospecies 10, respectively.
Relationship between PCR/ESI-MS and PFGE genotyping. A comparison of the classifications obtained by PCR/ESI-MS typing and PFGE is shown in Fig. 5. Dual classification was available for 200 isolates, mapped to 57 pies representing the number of sequence types; pies were then sliced to reflect the classification of these isolates into the 66 observed PFGE types. Fifty-two sequence types and PFGE types were clearly correlated and provided a consistent classification for 164 isolates (82% of the total evaluated [green in Fig. 5]). Twenty-one isolates were further discriminated by PFGE (Fig. 5, yellow). In particular, isolates belonging to ST14 were almost equally split into two PFGE types regardless of origin, suggesting that the PCR/ESI-MS genotyping method could be further improved for higher resolution. Conversely, five isolates were resolved by PCR/ESI-MS and not by PFGE (Fig. 5, blue pies) indicating that strain resolution by PFGE is also not exhaustive. For example, PFGE type 23 was composed of the closely related types ST28 and ST38. Isolates OIF123 and OIF129, characterized by PCR/ESI-MS as non-baumannii Acinetobacter species, shared PFGE types with A. baumannii isolates belonging to ST11 and ST30, respectively. PFGE and the PCR/ESI-MS genotyping methods were not in agreement for the 10 remaining isolates (Fig. 5, red).
DISCUSSION
Acinetobacter baumannii is a pathogen that has been associated with hospital outbreaks worldwide (6, 7, 9, 11, 18, 28, 33). This organism is particularly problematic due to the large number of multidrug-resistant strains, including the pan-European clone types I, II, and III, that have become endemic in hospital settings (12, 41). Recently, infections by Acinetobacter baumannii have resulted in critical complications for troops injured in Iraq and Afghanistan (47). The injured were treated in a variety of locations in the field and in European hospitals. As part of a public health investigation to identify the possible source(s) of this outbreak, to determine the likely mode(s) of transmission, and to assist in generating and implementing infection control countermeasures, we set out to develop a rapid Acinetobacter baumannii strain typing assay. The assay we developed is similar to traditional MLST strain typing, but analysis of PCR amplicons is performed with ESI-MS rather than standard DNA sequencing. The base composition of the PCR amplicons is determined from the masses of the PCR amplicons and provides enough information to type the strains. Previously we developed a similar assay for the rapid strain typing of respiratory pathogens (15, 16).
To develop the A. baumannii typing assay, we assembled a collection of 267 diverse isolates. Included in this collection were 216 isolates from U.S. and Iraqi patients involved in an outbreak in U.S. military hospitals associated with OIF, 23 isolates containing representatives of the endemic pan-European clones types I, II, and III, and a diverse collection of commercially available clones representing Acinetobacter baumannii and other Acinetobacter species.
Analysis of the collection of 216 Acinetobacter isolates from U.S. and Iraqi patients treated in U.S. military hospitals identified 31 (14.4%) A. baumannii isolates that were indistinguishable from the European reference strains that are plaguing European hospitals. Additionally, 80 OIF isolates (37%) differed from the endemic European reference strains by minor variations at a single locus, suggesting a relationship with the European endemic strains. The remaining 105 isolates represented more diverse strains.
Twenty-seven OIF Acinetobacter isolates (12.5%) were found to represent at least seven additional Acinetobacter species. Two of these were represented by multiple isolates and displayed intraspecies allelic variation; we were able to identify these two species as Acinetobacter genomospecies 13TU and Acinetobacter genomospecies 3 through comparison with relevant reference strains. Interestingly, these results mirror a recent study that identified the same two species amid infections by A. baumannii in hospital settings (10).
Three OIF isolates did not match any of our reference species and may represent novel Acinetobacter species. Due to sequence variation of the non-baumannii Acinetobacter isolates, we were not able to generate PCR/ESI-MS or sequencing PCR amplicons for these isolates from all primer pairs. Based upon the efp sequence and PCR/ESI-MS analyses from the other loci, we determined that these isolates were not Acinetobacter baumannii and did not match any of the 18 representatives of diverse Acinetobacter species tested. The unexpected diversity of these isolates highlights the potential of the PCR/ESI-MS approach to detect and strain type de novo infections from other Acinetobacter species.
Sequence analysis of the entire strain collection, including the European reference strains and the commercially available strains, identified 48 unique A. baumannii strain types. Interestingly, only one of the nine A. baumannii reference strains from the ATCC collection (ATCC 9955, ST9) was represented in the OIF isolates, suggesting that the bulk of the OIF sequences are distinct from the strains archived at the ATCC.
Base composition, though not as information rich as sequence data, has nearly the same resolving power for Acinetobacter baumannii strain typing as sequencing. By selecting primer pairs targeting nine information-rich regions of six loci, we were able to resolve by PCR/ESI-MS 47 of the 48 Acinetobacter baumannii strain types identified by sequencing 1,679 nucleotides from the six loci. The only case in which PCR/ESI-MS analysis did not resolve the strain type involved the two mutations (C to T and T to C) that distinguish isolate LUH6011 from the other clone III isolates: these mutations are mutually compensating, resulting in no net change in the PCR amplicon mass. Additional primers specifically targeting this isolate would bring no value to the resolution of other types, as the use of these nine primer pairs had already maximized the average number of mutations between all characterized types.
Our sequence analysis showed that the efp gene was not useful for discrimination of Acinetobacter baumannii isolates (Fig. 1), the initial goal of this study. However, the efp gene was particularly useful for discriminating Acinetobacter strains at the species level. The incorporation of the efp primer pair expanded the discrimination power of the PCR/ESI-MS Acinetobacter baumannii MLST assay to include the identification and typing of other Acinetobacter species.
In summary, we have used an A. baumannii MLST-like strain typing assay based on PCR/ESI-MS of nine PCR amplicons to identify 47 A. baumannii strain types. Fifteen additional types represented the related Acinetobacter genomospecies 13TU and Acinetobacter genomospecies 3. Using a portion of the efp locus, we distinguished at least 16 additional Acinetobacter species. The PCR/ESI-MS strain typing method is both rapid and low cost and can be performed in an automated modality, enabling the rapid surveillance of Acinetobacter spp. in a variety of settings. Use of this A. baumannii typing assay identified a genetic relationship between the endemic European isolates and many of the isolates found in patients and in military hospitals treating casualties from OIF. Further analysis of the OIF isolates and their locations of collection should provide better understanding of the origins of these infections and will ultimately improve infection control and prevention measures.
ACKNOWLEDGMENTS
We acknowledge DARPA and the CDC for financial support.
We thank Kyle Petersen for contributing Acinetobacter isolates from patients under his care on the U.S. Navy ship (hospital) Comfort while deployed in the Persian Gulf. We also acknowledge Amy Summers, of the Walter Reed Army Medical Center, for technical support in culturing and reisolation of strains used in this study and Lenie Dijkshoorn for providing reference sequences of Acinetobacter sp. strain 13TU.
FOOTNOTES
Corresponding author. Mailing address: Ibis Biosciences, Isis Pharmaceuticals, Inc., 1891 Rutherford Road, Carlsbad, CA 92008. Phone: (760) 603-3892. Fax: (760) 603-4653. E-mail: meshoo@isisph.com.
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