Quantitative Real-Time PCR for Detection of Acinetobacter baumannii Colonization in the Hospital Environment

Abstract

A real-time PCR assay was developed for detecting the presence of Acinetobacter baumannii on hospital equipment and compared to conventional bacterial culture using 100 hospital environmental samples. The real-time PCR detected contaminated surfaces in 4 h with high sensitivity (100%) compared to conventional culture. Thirty-eight percent of samples were positive by real-time PCR and negative by bacterial culture (false positives), possibly indicating the widespread presence of bacterial DNA that is not associated with viable bacteria. N osocomial infections caused by drug-resistant bacteria represent an important clinical challenge. Acinetobacter baumannii has become one of the most problematic causative agents of nosocomial infections due to its remarkable ability to survive on hospital surfaces and acquire antibiotic resistance, resulting in the global emergence of multidrug-resistant strains with resistance to multiple antibiotic classes (5). A. baumannii has been especially problematic in critically ill patients in the intensive care setting, as it is an important cause of ventilator-associated pneumonia and bacteremia. In this context, patients are exposed to A. baumannii via contact with contaminated hospital equipment or by contact with hospital personnel carrying the bacteria. A number of studies have demonstrated widespread contamination with A. baumannii on hospital environmental surfaces, most notably in intensive care units (ICUs) (1,4,8,9).Environmental surveillance protocols have been employed for the identification of hospital equipment colonized by A. baumannii so that appropriate decontamination procedures can be carried out (1,4,8,9). Since these surveillance methods employ conventional bacterial culture to determine the presence of A. baumannii, definitive species identification can require between 24 and 48 h. Nucleic acid-based tests, such as real-time PCR, have been employed for the identification of numerous bacterial pathogens (2); however, to our knowledge this technique has not been applied to identifying contaminated hospital equipment. The objective of the present study was to develop a real-time PCR for identifying hospital surfaces colonized by A. baumannii.A real-time PCR assay was developed using TaqMan chemistry for the amplification of nucleotides 774 to 859 of the outer membrane protein A gene (ompA; accession number AY485227). The ompA gene was chosen because it is present in all sequenced genomes of A. baumannii available in the public domain (as of March 2010), and the sequences chosen for the primers and probe correspond to regions highly conserved between published A. baumannii ompA sequences (100% sequence identity). The primers OmpA Forward (5=-TCTTGGTGGTCACTTGAAGC-3=) and Ompa Reverse (5=-ACTCTTGTGGTTGTGGAGCA-3=) and the probe (5=-AAGTTGCTCCAGTTGAACCAACTCCA-3=), 5= labeled with 6-carboxyfluorescein and the 3= labeled with 6-carboxytetramethylrhodamine, were used. A quantification standard, pGEM-ompA, was constructed by inserting the ompA gene from the ATCC 19606 strain...

Molecular-based detection of potentially pathogenic bacteria in membrane bioreactor (MBR) systems treating municipal wastewater: a case study

“…dPCR was performed using primers targeting Acinetobacter baumannii (McConnell et al 2012) ( ompA ), Klebsiella pneumoniae (Lee et al 2006) ( phoE ), and Pseudomonas aeruginosa (Lee et al 2006) ( regA ). Relative gene abundances were normalized per liter of sample.…”

Multiplex identification of sepsis‐causing Gram‐negative pathogens from the plasma of infected blood

“…For precise multiplex detection of the major sepsis‐causing Gram‐negative pathogens, we developed species‐specific MLPA probes by modifying known species‐specific sequences which were confirmed by previous studies ( uidA and lacY for E. coli , ompA for A. baumannii , phoE for K. pneumoniae , and ecfX for P. aeruginosa ) (Table ). Singleplex MLPA reactions were performed to assess the specificity of each probe set.…”

Detection of Bacterial 16S rRNA and Identification of Four Clinically Important Bacteria by Real-Time PCR

“…OmpA was included as a potential target as this gene has been used as a target for A. baumannii -specific primers in previous publications [11]. However, though ompA was identified as a potential target using our algorithm, sequence variation among A. baumannii , A. nosocomialis and A. pitii , as well as variation in the protein length, resulted in this gene being flagged as unsuitable as an Acinetobacter -specific target.…”