Phenotypic and Genotypic Characterization of Methicillin-Resistant Staphylococcus aureus (MRSA) and Methicillin-Susceptible Staphylococcus aureus (MSSA) from Different Sources in China

Introduction

S taphylococcus aureus is a robust pathogen that causes human diseases by producing and secreting an array of virulence factors such as coagulase, hemolysin, enterotoxins, biofilm genes, and immune system evasion genes (Tristan et al., 2007; Ellis, 2008). S. aureus infection is facilitated by its propensity to develop resistance to multiple antibiotics; methicillin-resistant S. aureus (MRSA) is the most frequently identified antibiotic-resistant pathogen in humans. Resistance to methicillin and other ß-lactam antibiotics is conferred by the mecA gene, which is situated on the mobile genomic island staphylococcal cassette chromosome mec (SCCmec). There are currently two types of MRSA recognized: hospital-acquired MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA). CA-MRSA infections are contracted outside of the hospital setting and account for a recent rise in the incidence of MRSA infections since the late 1990s. CA-MRSA differs from HA-MRSA with respect to epidemiology, microbiology, and clinical manifestations (David and Daum, 2010). HA-MRSA strains carry a large SCCmec gene that is subgrouped into SCCmec types I, II, or III, whereas CA-MRSA isolates carry smaller SCCmec elements such as SCCmec type IV or type V (Pantosti and Venditti, 2009; David and Daum, 2010).

Animals such as domestic pets, livestock (particularly pigs), and wild birds have been identified as carriers of MRSA (mainly CA-MRSA) in regions that harbor common human MRSA strains (David and Daum, 2010). However, until recently, there have been few reports on the MRSA status of animal reservoirs in China (David and Daum, 2010; Wanfang Data, 2012).

Antibiotic resistance is a global problem. Under the selective pressure of excessive antibiotic use, local evolution of multidrug resistance (MDR) bacteria (resistance to three or more antibiotics) occurs through horizontal gene transfer, leading to the transmission of genes such as the ß-lactamase genes between bacterial species and strains (Hawkey and Jones, 2009). MRSA, particularly HA-MRSA, is usually associated with the expression of MDR genes including non-ß-lactam resistance genes encoding resistance to aminoglycosides, macrolides, and tetracyclines (David and Daum, 2010).

In this study, 261 strains from different sources such as human, animal, food, and environmental sources in China were characterized using phenotypic and molecular methods. All strains were tested for the presence and type of SCCmec elements, the presence of non-ß-lactam antibiotic resistance genes, and antibiotic susceptibility.

Materials and Methods

Results

Discussion

From 2001 to 2010, there have been reports regarding SCCmec typing of MRSA isolated from hospital settings in China. In all 1260 MRSA strains reported from Beijing (Wang et al., 2009; Zhao et al., 2011), Hunan (Pan, 2009), Shanghai (Zhang, 2009), Sichuan (Li et al., 2008), Zhejiang (Yu et al., 2006), Anhui (Ji et al., 2007), Tianjin and Wuhan (Kong et al., 2009), Shenzhen (Xiao et al., 2007), Ningxia (Zhang et al., 2011), Liaoning (Pan et al., 2011), and Guangdong (Ma et al., 2008), SCCmec type III accounted for 87.30% (1100/1260) cases followed by type II, type IV, type V, and type I, accounting for 6.59% (83/1260), 2.46% (31/1260), 0.79% (10/1260), and 0.40% (5/1260) of cases, respectively; 2.46% (31/1260) of MRSA strains were untypable. Our results and reports above indicate that HA-MRSA but not CA-MRSA remains the major pathogenic bacterium in the clinical setting; however, infections caused by CA-MRSA have been increasing in recent years. Until recently, there have been few reports regarding the epidemiology of CA-MRSA in China (David and Daum, 2010; Wanfang Data, 2012).

In Japan, Pakistan and India, and Korea, most of the MRSA strains that were isolated from hospitals were HA-MRSA (mainly type III or II), but infections by CA-MRSA have also been increasing in these countries in recent years (Ko et al., 2005; Ohkura et al., 2009; Park et al., 2009; Shabir et al., 2010; Yamada et al., 2011). HA-MRSA strains have been isolated largely from people who are exposed to the healthcare setting; inpatient hospital patients tend to be older and tend to have one or more comorbidities, which increases the chances of developing MRSA infections (Enright et al., 2002; Millar et al., 2007; Carvalho et al., 2010; David and Daum, 2010; Santos et al., 2010). The nosocomial environment also facilitates the development of HA-MRSA through the selective pressure of antibiotics.

CA-MRSA has emerged worldwide as a cause of infections among patients without the risk factors typically associated with hospital patients (David and Daum, 2010). In the current study, CA-MRSA strains were isolated from both human and animal sources including cows and pigs (as sources also including dogs and chickens). The percentage of CA-MRSA strains from animals was significantly higher than that from humans (19.67% versus 5.79%, p<0.01); also, the percentage of CA-MRSA strains from the porcine hosts and the cow hosts was significantly higher than that from humans, respectively (25.81% versus 5.79%, 15.38% versus 5.79%, p<0.01, respectively). Our results support the idea that animals function as reservoirs of CA-MRSA and source of human MRSA. There have been sporadic reports of CA-MRSA being isolated from animals in China: six CA-MRSA strains (five SCCmec type IV and one that could not be identified but belonged to ST9, the same ST as SCCmec type IV) out of 54 total S. aureus strains were isolated from diseased cows in five provinces (Wang et al., 2011); another 14 CA-MRSA strains (all of which belonged to SCCmec type IV) out of 83 total S. aureus strains were isolated from pigs in Shanxi province (Wang et al., 2010). Food animals, particularly pigs and dairy cows, have been found to be important sources of CA-MRSA infections in humans (Lee, 2003; Thorberg et al., 2006; Juhasz-Kaszanyitzky et al., 2007; Tiwari and Tiwari, 2007; van Loo et al., 2007; Khanna et al., 2008; van Rijen et al., 2008; Kock et al., 2009; Lozano et al., 2011). In our study, no MRSA strains were isolated from food or environmental sources; however, food and environmental sources may also serve as important CA-MRSA reservoirs because S. aureus has been shown to survive for long periods of time on inanimate objects (Simoes et al., 2011; Uhlemann et al., 2011). Some studies have reported about human carriers of CA-MRSA strains, but they could not identify the source of the pathogen (Chatterjee et al., 2009; David and Daum, 2010; Denis et al., 2009; Lamaro-Cardoso et al., 2009; Chen et al., 2011). Even so, animal reservoirs have increasingly been considered to represent an important source of human MRSA acquisition. Therefore, hospitalized patients may become colonized with both CA-MRSA and HA-MRSA as the community reservoir of CA-MRSA continues to increase (D'Agata et al., 2010).

The regional resistant genes combinations are more helpful for analyzing the resistance genotypes and phenotypes of S. aureus (Udo and Dashti, 2000). Most of MRSA strains and a part of MSSA stains harbored unique combinations of aac(6′)/aph(2′′), aph(3′)-III, and ant (4′,4′′), ermA, ermC, and mrsA, tetM, and tetK genes. Antibiotic resistance genes were detected more frequently in HA-MRSA strains than in CA-MRSA strains (p<0.01), and also more frequently in MRSA strains than in MSSA strains (p<0.01). Almost all of HA-MRSA strains carried three genes, aac(6′)/aph(2′′), ermA, tetM, conferring resistant to kanamycin and amikacin, erythromycin, tetracycline, the important antibiotics in clinic. CA-MRSA isolates have typically been susceptible to most non-ß-lactam antimicrobial drugs (David and Daum, 2010), while in this study, all CA-MRSA strains harbored different non-ß-lactam-resistant genes and are resistant to the majority of the relative antibiotics (Table 1). The results show that CA-MRSA strains might acquire different resistance genes gradually, depending on the selective pressure of antibiotics in different regions or environments.

Some strains harbored non-ß-lactamase genes but were susceptible to the relative antibiotics, and these genes were detected mainly in non-MDR MSSA strains and less in HA-MRSA strains. Other studies have also reported similar findings (Vanhoof et al., 1994; Udo and Dashti, 2000). The phenomenon in this study also confirmed that the resistance genes are acquired on the selective pressure exerted by antibiotics and antiseptics in the environment; these strains will express their resistance at the exposure of relative antibiotics at a later date (Udo and Dashti, 2000).

We observed that no MRSA strains were resistant to vancomycin and teicoplanin, and most MRSA strains were susceptible to chloramphenicol and rifampicin in this study. Certain MRSA strains have been reported to demonstrate resistance to vancomycin and teicoplanin in some countries (David and Daum, 2010). Almost all of the HA-MRSA strains were resistant to the non-ß-lactam antibiotics gentamicin, erythromycin, and tetracycline, while approximately half of the CA-MRSA strains were resistant to the same antibiotics. Our results show that HA-MRSA strains were significantly (p<0.01) more resistant to different antibiotics than CA-MSSA strains. The results also show that not only HA-MRSA but also CA-MRSA strains are developing resistance to more antibiotic agents. Information about MRSA and MSSA antibiotic profiles in this study is also important for the effective treatment of regional S. aureus infections. Statistical analysis revealed that the association between the antibiotics in MRSA strains and MSSA strains was highly significant (p<0.01). As the MDR MSSA strains were higher and these strains were obtained from animal, food, and human sources, the results suggest that drug-resistant bacteria or drug-resistance genes could be transferred from animals to food to humans, and that transfer in this manner could lead to the proliferation of increasingly drug-resistant strains (Aarestrup and Wegener, 1999). MDR MSSA strains obtained from food, animal, and human sources might also become an emerging problem for human medicine in terms of the spread of resistance, which warrants further investigation.

Conclusions

In conclusion, HA-MRSA remains a large problem in hospitals in China, whereas CA-MRSA is not yet endemic but appears to be increasing in prevalence in China. Animals such as pigs, cows, and household pets appear to serve as major reservoirs for CA-MRSA, which is often transmitted from animals to humans by direct contact. MRSA and MDR MSSA strains harbored many resistance genes in many unique combinations and thus displayed resistance to several antibiotics. Based on this study, our results support the value of increasing the surveillance of MRSA and implementing MRSA control measures in animals and humans in China.