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J Clin Microbiol. Nov 2010; 48(11): 4333–4336.
Published online Aug 25, 2010. doi:  10.1128/JCM.01585-10
PMCID: PMC3020819

Genotypic and Phenotypic Correlations of Multidrug-Resistant Acinetobacter baumannii-A. calcoaceticus Complex Strains Isolated from Patients at the National Naval Medical Center[down-pointing small open triangle]

Xiao-Zhe Huang*
Division of Bacterial and Rickettsial Diseases
Walter Reed Army Institute of Research
Silver Spring, Maryland
Jonathan G. Frye
Bacterial Epidemiology and Antimicrobial
Resistance Research Unit
Agriculture Research Service
U.S. Department of Agriculture
Athens, Georgia
Mohamad A. Chahine and Dana M. Cash
Division of Bacterial and Rickettsial Diseases
Walter Reed Army Institute of Research
Silver Spring, Maryland
Melissa G. Barber
Walter Reed Army Medical Center
Washington, DC
Britta S. Babel, Matthew R. Kasper, and Timothy J. Whitman
National Naval Medical Center
Bethesda, Maryland
Luther E. Lindler
Chemical and Biological Division
Science and Technology Directorate
Department of Homeland Security
Washington, DC

The Acinetobacter baumannii-Acinetobacter calcoaceticus complex (ABC) has emerged as a significant opportunistic pathogen that is often the cause of nosocomial outbreaks. Most ABC isolates are resistant to almost all currently available antibacterial agents (7). With increased concern about antimicrobial resistance (AR) at both military treatment facilities and civilian hospitals, a study to better understand the correlation of the ABC genotype and AR phonotype will provide important information to benefit diagnosis and epidemiological surveillance.

In this study, 102 ABC isolates from 59 wounded soldiers returning from Iraq and treated at the National Naval Medical Center (NNMC) from June 2006 through December 2006 were characterized by phenotype and genotype to identify clones in this population and to determine genetic relationships. Pulse-field gel electrophoresis (PFGE) revealed 23 PFGE types (PFTs) based on ≥90% similarity using Dice coefficients with BioNumerics software (Applied Maths, Kortrijk, Belgium). Eighty percent of the isolates belonged to six dominant PFTs, while the remaining isolates had limited similarity to other isolates (Fig. (Fig.1).1). PFGE patterns showed correlation with overall antibiogram results (Fig. (Fig.11 and Table Table1).1). For example, PFT 1, the most dominant cluster, correlated well with antimicrobial susceptibility testing type (ASTT) patterns, with 83% representation of ASTT 13 in this PFT. Similar results were also seen in other PFTs in Fig. Fig.1.1. However, there were also examples of isolates with varied ASTTs clustered in the same PFT (PFT 5 and PFT 11), which may be explained by differences in the antibiotic resistance gene expression of individual strains in AST assays or independent genetic mechanisms such as loss of plasmid-mediated resistance (1). On the other hand, approximately 10% of the isolates with similar AST patterns (e.g., ASTTs 13, 15, etc.) were found in different PFTs (Fig. (Fig.1),1), implying that horizontal gene transfer may have played a role in the dissemination of antibiotic resistance, as described in earlier reports (3, 5).

FIG. 1.
Comparison of genotypic and phenotypic features of ABC strains isolated at the NNMC. Superscript numbers: 1, identification (ID) number; 2, AST result for imipenem (R, resistance; S, susceptible; I, intermediate); 3, PCR results with OXA27/23 primers ...
TABLE 1.
MICs of representatives of ASTTs and subtypes of NNMC ABC isolatesa

To investigate the genetic basis of drug resistance, a DNA microarray (2, 9) was used to analyze specific isolates in this study. Four representative isolates selected from each of the three most dominant PFTs (PFT 1, PFT 5, and PFT 11), one from a minor PFT (PFT 17), and the three reference isolates from the Walter Reed Army Medical Center (WRAMC) were analyzed using the DNA microarray (n = 8). Eleven aminoglycoside resistance genes, four β-lactamase genes, two quaternary ammonium resistance genes, three sulfonamide resistance genes, three tetracycline resistance genes, and five transfer-associated genes were detected in the isolates tested. Only isolates hybridizing with a probe for a blaOXA gene were resistant to imipenem. PCR and sequencing analysis of these isolates determined that the imipenem resistance gene was a globally spread blaOXA-23 gene (4). Further PCR analysis showed that 27% of the 102 ABC isolates were blaOXA-23 positive. Drug susceptibility tests and the PCR test correlated well, indicating that 93% of the imipenem-resistant isolates carried the blaOXA-23 locus. Isolates harboring blaOXA-23 tended to cluster into two distinct PFTs, with 17 isolates belonging to PFT 5 and 10 isolates belonging to PFT 11. Our study found there were certain correlations between the genotypes and AR phenotypes of the ABC bacteria studied, which agreed with previous limited reports (6, 8). The combination of molecular characterizations and classical AST provided precise multidrug resistance information that can benefit diagnosis, surveillance, epidemiologic analysis, and preventive strategies for multidrug-resistant ABC infections.

Acknowledgments

We thank Emil Lesho at the Walter Reed Army Institute of Research (WRAIR) and Rosa Ergas at the NNMC for providing some background information on the isolates. James Snyder at the University of Louisville Hospital advised on the interpretation of AST results. Jennifer Turpin provided technical assistance with microarray tests.

This work was funded by the DoD-Global Emerging Infectious Disease Surveillance.

The findings and opinions expressed herein are ours and do not necessarily reflect the official views of the WRAIR, the U.S. Army, or the Department of Defense.

Footnotes

[down-pointing small open triangle]Published ahead of print on 25 August 2010

REFERENCES

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