Development of real-time PCR systems based on SYBR Green for the specific detection and quantification of Klebsiella pneumoniae in infant formula

Abstract

Klebsiella pneumoniae is an emerging opportunistic pathogen that causes severe morbidity and mortality among the newborn and immunocompromised persons. Infant formula has been associated with the K. pneumoniae-related outbreaks in a newborn intensive care unit. In this study, a SYBR Green real-time PCR assay was developed for the rapid identification of K. pneumoniae from infant formulas. 142-bp phoE gene was selected for specificity, and two K. pneumoniae strains and 15 non-K. pneumoniae strains were evaluated. All K. pneumoniae strains tested were phoE-positive to the real-time PCR detection and no cross-reactivity was observed with non-K. pneumoniae strains. Sensitivity of the detections is 1 CFU/25 g infant formulas. And the assays can be accomplished within two business days including pre-enrichment. The results indicate that the method is efficient and can be employed to detect specifically K. pneumoniae from infant formulas with considerable sensitivity. In this study, we also constructed reference standards for quantification of K. pneumoniae by real-time PCR. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the reference standards, indicating that they can be used as quantitative detection of K. pneumoniae. This result is of special significance for applications of predicting the load of K. pneumoniae.

Introduction

After the public-health scandal of toxic milk in China, the government strengthened the supervision of the market to guarantee food safety, which initially involved melamine in infant formula, but then extended to other aspects, such as pathogenic microorganisms. Klebsiella pneumoniae is a well-recognized hospital-infection pathogen. It is responsible for a significant proportion of hospital-acquired infections including septicemias, pneumonia, urinary tract infections, and soft tissue infections especially in the immunocompromised hosts such as the neonate (Alcantar-Curiel et al., 2004, Archana Gupta, 2002, Jerassy et al., 2006). K. pneumoniae can cause severe morbidity and mortality especially among the newborn (Bingen, 1993, Casolari et al., 2005, Coovadia et al., 1992). In recent years, K. pneumoniae have frequently been reported as the cause of outbreaks involving newborn infants in neonatal intensive care units (Fangyou et al., 2009, Randrianirina et al., 2009, Jerassy et al., 2006, Casolari et al., 2005, Wachino et al., 2004, Gastmeier et al., 2003, Lebessi et al., 2002, Martinez-Aguilar et al., 2001, Macrae et al., 2001, Roilides et al., 2000, Van der Zwet et al., 1999, Al-Rabea et al., 1998, Villari et al., 1998, Banerjee et al., 1993, Reish et al., 1993, Arredondo-Garcia et al., 1992, Denny et al., 1986, Donowitz et al., 1981, Hill et al., 1974, Mayhall et al., 1980, Saravolatz et al., 1984, Sveningsen et al., 1984). K. pneumoniae have also been found in powdered infant formula (Carneiro et al., 2003, Muytjens et al., 1988). As potential powdered infant formula-borne pathogens, K. pneumoniae was classified in the hazard identification category “B” by FAO and WHO (FAO/WHO, 2004). Comparison to other enterobacteriaceae in the category “B”, K. pneumoniae was the most important pathogen for its severe morbidity and mortality (Liu et al., 2008).

In order to minimize the hazards caused by K. pneumoniae and limit the spread of these pathogens, it is crucial to develop a rapid, specific and sensitive method for early detection of this bacterium in infant formula. The conventional methods are laborious and time-consuming; they involve isolating in pure culture and performing biochemical tests. These methods generally take 5–7 days to obtain the definitive results (FAO, 1994). Recently, real-time PCR has emerged as a valuable tool for the rapid detection of microorganisms in different samples. Real-time PCR has similar sensitivities as classical methods, but consumes less time and labor intensive. Another advantage of this method is that no post-PCR steps are required, thus avoiding the possibility of cross contamination due to PCR products, eliminates the occurrence of false positive (Ginzinger, 2002).

In this study, we describe a real-time PCR assay with specific primers targeting the phoE gene for the detection of K. pneumoniae from infant formula samples. The assay also provides specific and sensitive quantification of K. pneumoniae DNA.