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Although Staphylococcus aureus (S. aureus) has the highest pathogenic potential among the many species of staphylococci, coagulase-negative staphylococci (CoNS) have become an increasing concern in human and veterinary medicine (Rice 2006; Fessler et al. 2010). CoNS can be associated with a variety of animal and human diseases, such as suppurative disease, arthritis and urinary tract infection in animals (Lee 2003) and skin and soft tissue infections and bacteremia in humans (Martins and Cunha Mde 2007). Animals are common reservoir of Staphylococcus and can carry CoNS on their skin, noses, upper alimentary and urogenital tract and intestinal tract and thus may transmit the bacteria to human by animal handling (Werckenthin et al. 2001). CoNS carried by food animals can also contaminate the animal products and enter the food chain, thus posing potential threat to food consumers and handlers.
More importantly, methicillin-resistant CoNS (MRCoNS) have been isolated worldwide from food animals (Kawano et al. 1996; Fessler et al. 2010; Haenni et al. 2011), including pigs, cows, calves and chicken and therefore may compromise the treatment if animal or human infection occurs. Interestingly, resistance to non-β-lactam antimicrobials, such as erythromycin, tetracycline, clindamycin, ciprofloxacin and sulfamethoxazole/trimethoprim, is common in MRCoNS (Simeoni et al. 2008; Huber et al. 2011). A recent study from Switzerland revealed 48·2% of MRCoNS from livestock, and high percentages of CoNS were also resistant to non-β-lactam antimicrobials, suggesting that multidrug-resistant CoNS may become an emerging problem for veterinary medicine and public health (Huber et al. 2011). However, most studies on CoNS in animals have been performed in Europe. There are limited data on CoNS in US food animals as well as resistance of CoNS to non-β-lactam antimicrobials.
In addition to mec(A) that is carried by a mobile genetic element, staphylococcal cassette chromosome mec (SCCmec), and confers broad-spectrum β-lactam resistance, various antimicrobial resistance genes have been identified in CoNS from food production environment. erm(A), erm(B), erm(C), vga(B), tet(K) and tet(M) have been found in CoNS resistant to the macrolide–lincosamide–streptogramin B (MLSb) antimicrobials and tetracyclines (Aarestrup et al. 2000; Simjee et al. 2007; Graham et al. 2009), the antimicrobials commonly used in animal production in the United States. As many of these genes are carried on extra-chromosomal elements, including conjugative plasmids and transposons (Aarestrup et al. 2000; Schmitz et al. 2001; Khan et al. 2002; Simjee et al. 2007), CoNS strains may become an important reservoir of antimicrobial resistance genes that have potential to transfer to other commensal bacteria or even pathogenic ones.
The present study was aimed to understand CoNS of animal origin as a reservoir of multidrug resistance. We examined the antimicrobial susceptibility phenotypes and genotypes of CoNS from agricultural animals and estimated the potential of tetracycline resistance gene transfer by conjugation.
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- Materials and methods
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Most studies on antimicrobial-resistant Staphylococcus in agriculture have focused on Staph. aureus from food or food-producing animals, whereas less research effort has been put on CoNS, a group of staphylococci that are believed to be a larger reservoir of antimicrobial resistance genes (Lee 2003; O'Mahony et al. 2005; Juhasz-Kaszanyitzky et al. 2007). The recovery of 47 multidrug-resistant CoNS (54%) from food animals and the observation of resistance to macrolides, tetracyclines and streptogramin, in addition to β-lactam resistance, suggest food animals as important reservoir of antimicrobial-resistant CoNS.
The four Staphylococcal species, Staph. lentus, Staph. sciuri, Staph. xylosus and Staph. haemolyticus, identified in this study are commonly associated with farm animals (Aarestrup and Schwarz 2006). The high prevalence of resistance to tetracycline, erythromycin and clindamycin was not surprising as compared to previous studies on CoNS in poultry litter in the United States (Simjee et al. 2007) and MRCoNS from various sources, including animals, meat and contact persons, in Switzerland (Huber et al. 2011). When comparing the three studies, we found tetracycline resistance (67·8%) predominated in our study and so did erythromycin resistance (71%) in the Simjee study, whereas no marked difference was observed in the Swiss study in terms of the prevalence of resistance to erythromycin, tetracycline and clindamycin, ranging from 43·7 to 49% (Huber et al. 2011). This may be an indication of common use of tetracyclines and macrolides in animal production in the United States. Recovery of Q/D-resistant CoNS in both of the US studies, with prevalence of 14·9% and 13%, respectively, suggests a potential linkage between streptogramin usage on farms and Q/D resistance in this country, although comparison between the US data and data from Switzerland was not possible due to the exclusion of Q/D in the Swiss study. As Q/D can be used to treat methicillin-resistant Staphylococcal infection in humans (Hamel et al. 2008), our study suggests a need to monitor the Q/D resistance in CoNS of animal origin and to understand the potential of Q/D-resistant CoNS to cause human disease.
Because all CoNS included in this study were potentially resistant to β-lactam, it was not unexpected that the isolates were resistant to at least one β-lactam antimicrobial. The recovery of high percentages of multidrug-resistant CoNS from most animal categories suggests that animal commensal bacteria are important reservoir of antimicrobial resistance phenotypes. Because animal intestinal environment provides an optimal condition for antimicrobial resistance genes to transfer from commensals to pathogens (Marshall et al. 2009), gene dissemination across species or even genus borders is expected to be common in animal hosts, and the extent of antimicrobial resistance can thus be amplified substantially. Multidrug resistance phenotypes were commonly seen in the top three animals carrying CoNS and the prevalence was 37%, 60% and 61·5%, from cattle, sheep and goats, respectively, which again is an indication of widespread distribution of multidrug resistance in agriculture, although farm variation in agricultural practices and level of antimicrobial exposure of bacteria could not be excluded.
The observation of staphylococcal species variation in multidrug resistance is consistent with findings by Huber et al. (2011) that resistance of CoNS to erythromycin, tetracycline, clindamycin, ciprofloxacin, sulfamethoxazole/trimethoprim and gentamicin was more common in Staph. haemolyticus, Staph. epidermidis and Staph. sciuri than that in other species, with Staph. haemolyticus having the highest percentage of non-β-lactam resistance. Another study also found 4 multidrug-resistant Staph. haemolyticus of six total mec(A)-positive Staphylococci from clinical animals (van Duijkeren et al. 2004). Together with our results that all nine Staph. haemolyticus vs only one of 12 Staph. xylosus were multidrug resistant, it is reasonable to assume that species variation exists in Staphylococcus as to their antimicrobial resistance. Staphylococcus haemolyticus in animals may have stronger public health significance, considering that it is frequently involved in human clinical cases (van Duijkeren et al. 2004; Huber et al. 2011).
erm(A) and erm(C) were commonly found in erythromycin-resistant Staphylococci in this study, which is in agreement with previous reports that they were the predominant erythromycin resistance genes in Staphylococcus isolated from various sources (Aarestrup et al. 2000; Khan et al. 2002; Simjee et al. 2007; Graham et al. 2009). Unlike erm(A) and erm(C), erm(B) is less common in Staphylococcus than in Enterococcus and Streptococcus. However, erm(B) was identified in seven erythromycin-resistant CoNS in our study, in contrast to its absence in previous studies (Aarestrup et al. 2000; Simjee et al. 2007; Graham et al. 2009), suggesting genetic diversity in erythromycin resistance genes in Staphylococci in different geographical locations. As erm(B) can be carried on plasmids, the chromosome and on transposons (Khan et al. 2002), future examination of the genetic background of erm(B) in these isolates would help assess the potential of its dissemination in Staphylococcus in animals. Our finding that 13 of 32 erythromycin-resistant isolates had no resistance genes identified was not surprising as compared to previous reports (Simjee et al. 2007), although it could also be due to the limited number of genes tested.
As for streptogramin resistance genes, the vga(A) gene detected was confirmed by DNA sequencing as vga(A)LC. This evolutionary variant of vga(A) encodes for an ABC transporter and has been reported in Staphylococcus resistant to lincosamides and streptogramin (Novotna and Janata 2006; Qin et al. 2011). Failure to observe clindamycin resistance in some of our vga(A)LC-positive isolates can be explained by the different phenotypic methods used to determine clindamycin resistance because we used a broth microdilution method whereas agar dilution or disc diffusion was applied in previous studies. In fact, the clindamycin resistance level of Staph. haemolytics carrying vga(A)LC reported by Novotna et al. was not very high (Novotna and Janata 2006), so borderline resistance phenotypes would not be unexpected when using a different method. In addition, source of isolation may also have indication on antimicrobial resistance phenotypes. All previously reported Staphylocccus carrying vga(A)LC was from clinical settings where constant antimicrobial selective pressure maintains the resistance phenotypes more effectively than what the antimicrobial selection does in agriculture.
The two mechanisms of tetracycline resistance reported in Staphylococcus, ribosomal protection and active efflux, were identified in this study as evidenced by the presence of tet(M), encoding for ribosomal protection, in more than 60% of CoNS, and tet(K), encoding for an active efflux, in 45·7% of the isolates. Tn916, a conjugative transposon, which is often associated with tet(M), was detected in most tet(M)-positive isolates. The fact that nearly 17% of CoNS could transfer tet(M) to Enterococcus faecalis, together with Tn916, suggests that tetracycline resistance from Staphylococcus can transfer to other Gram-positive bacteria that have potential to cause human diseases. However, considering the high prevalence of Tn916 in our isolates, this conjugation rate was low, which might be due to the variation in resistance transfer to E. faecalis from different Staphylococcus species, especially when lineage variation in tetracycline resistance transfer has been observed from Staph. aureus strains to Staph. aureus recipients (de Vries et al. 2009). As antimicrobial resistance genes other than tet(M) have been identified on Tn916 (Fletcher and Daneo-Moore 1992; Del Grosso et al. 2004; Lancaster et al. 2004), transfer of additional antimicrobial resistance genes should also be expected via this conjugative transposon from a multidrug resistance reservoir.
In conclusion, our data indicate that CoNS in agricultural animals are an important reservoir of multidrug resistance in addition to the resistance to β-lactam antimicrobials and underline the importance of surveillance of multidrug-resistant CoNS in the food production environment. As our CoNS strains were all resistant to at least one β-lactam antimicrobial, further research is needed as to whether methicillin resistance predisposes CoNS to become multidrug resistant as compared to general CoNS, including the potential linkage, if any, between β-lactam resistance and other resistance phenotypes. Species variation exists in the prevalence of multidrug resistance in Staphylococci. Certain Staphylococcal species, such as Staph. haemolyticus, may have stronger potential to become multidrug resistant and thus require closer research and public health attention.