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An exploratory study of methicillin-resistant Staphylococcus aureus (MRSA) and SCCmec elements in bacteria along the Mexican border of south Texas was performed. Between September and December of 2008, 375 swabs of anterior nares were self-collected by students attending the University of Texas-Pan American (UTPA) and cultured for MRSA. Fifty seven bacterial isolates were kept for further analysis that included suspected MRSA and other SCCmec-containing bacteria. Isolates were examined for the presence of nuc, mecA, lukS-PV, and spa genes using PCR. SCCmec and spa typing were also performed. Seven S. aureus isolates were found of which six were classified as MRSA. SCCmec typing showed five of the six MRSA strains to be type IV, while one MRSA strain, and most of the non-S. aureus strains, were untypeable, producing results that were indicative of mixed SCCmec types. Five of the six MRSA strains contained known spa types (two of which corresponded to USA300 and one to USA600), while one strain had a novel spa type. Only one isolate, a USA300 MRSA, was positive for lukS-PV. Easy access by the Texas border community to antibiotics in Mexico without a prescription, and the strong partition in SCCmec types between MRSA and non-S. aureus bacteria suggest that this border region of Texas may be uniquely suited for the study of emerging SCCmec types, their horizontal transfer, and perhaps other aspects of antibiotic resistance in bacteria.
Staphylococcus aureus is a bacterium that is commonly isolated from the anterior nares of humans where it is considered a natural inhabitant. Pathogenically, S. aureus is characterized as not normally causing disease in man except when an ‘opportunity’, such as a cut or lowered immune system, presents itself, in which case the infection can normally be treated successfully with antibiotics. However, some strains of S. aureus have been shown to be more highly resistant to antibiotics, which makes infection difficult to treat and the strains more serious pathogens. Increased resistance to antibiotics has been shown to be due in part to the presence of a genomically located mobile DNA element referred to as the Staphylococcus Cassette Chromosome, which contains the mecA gene (SCCmec) . The mecA gene product is a modified penicillin-binding-protein (PBP2a), which confers resistance to the large group of beta-lactam antibiotics, such as methicillin . SCCmec also carries a gene coding for a recombinase that facilitates the movement of SCCmec to other bacteria that do not have the element. In some bacteria SCCmec has been found to also contain plasmids or other mobile DNA elements carrying diverse antibiotic resistance genes, thus expanding the range of antibiotic resistance beyond beta-lactams. When a strain of S. aureus acquires a copy of SCCmec, the bacterium is referred to as a methicillin-resistant S. aureus, or MRSA for short. Historically, MRSA have been associated primarily with health care facilities, such as hospitals, where antibiotics are commonly used and where SCCmec presumably has given S. aureus a fitness advantage. These strains of MRSA are referred to as healthcare-associated MRSA (HA-MRSA). More recently however, strains of MRSA have been isolated from apparently healthy individuals residing in the community, who had no apparent link to the healthcare system, and are referred to as community-associated MRSA (CA-MRSA) . CA-MRSA are generally characterized as demonstrating less antibiotic resistance but more virulence due to the presence of virulence factors such as the Panton-Valentine leukocidin (PVL), a cytotoxin associated with disease . For both healthcare and scientific reasons, there is much interest in understanding the level of diversity within the SCCmec element, and the geographic range of both the different strains of MRSA and the SCCmec elements they possess. Useful to such studies are a number of bacterial-typing methods that have been developed for both MRSA and SCCmec. In particular, sequence analysis of the different SCCmec elements has shown that most MRSA can be grouped into at least one of five established SCCmec types, where types I through III are typical of HA-MRSA and types IV and V of community-associated strains . More recently, SCCmec elements of types VI , VII , and VIII  have been proposed, along with reports of untypeable elements, raising the question of even more SCCmec types, and the need of a more standardized and comprehensive system for SCCmec typing . Similarly, it has been shown that the number and sequence of DNA repeats within the Short Sequence Repeat (SSR) region of the spa gene in S. aureus can be used to show phylogenetic relationships between different MRSA isolates .
Although MRSA and the SCCmec elements have been studied throughout the United States and the world, relatively little work has been undertaken along the U.S. border with Mexico. The Texas/Mexico border region represents an environment largely unique from the rest of the United States that is characterized by relatively high poverty, a largely Hispanic population, and relatively easy access by U.S. and Mexican citizens to antibiotics without a prescription [2, 17]. It is of public health and scientific importance, therefore, to determine if MRSA/SCCmec in the border region is evolving differently than elsewhere in the United States, and whether the uniquely transitional environment of the border region may promote the emergence of new, more serious strains of MRSA that have the potential to spread throughout the rest of the United States. To date, limited studies have been undertaken to study MRSA/SCCmec along the U.S./Mexico border, and those that have, have focused on strains obtained from a hospital setting [5, 14, 17]. Thus very little is currently known about MRSA, and their SCCmec elements, in communities located along the Texas border with Mexico. Herein, we report on an exploratory study of MRSA and SCCmec in a Texas border community in order to provide needed baseline data on MRSA and SCCmec elements in this important transitional region.
Between June and December of 2008, 375 swabs of the anterior nares were self-collected by student volunteers attending the University of Texas-Pan American (UTPA), located along the Texas border with Mexico. The student population at UTPA is comprised primarily of individuals of Hispanic descent living along the Texas middle Rio Grande Valley and across the border in Mexico. The study was approved by the UTPA Institutional Review Board and informed consent by students volunteering samples was obtained. Swabs were returned to their paper wrappers after collection then immediately taken to the laboratory and placed in 5 ml of Iso-Sensitest broth (Oxoid Ltd., Basingstoke, Hampshire, England) supplemented with 2.3% NaCl, 4 μg/ml cefoxitin, 8 μg/ml colistin, 8 μg/ml of aztreonam . Inoculated broths were incubated for 48 h at 33–35°C, then cultured on Mannitol Salt Agar (MSA, Accumedia Manufacturing Inc., Lansing, Michigan) for 24 h. Colonies showing a yellow to gold color characteristic of S. aureus, as well as representatives of other diverse colony phenotypes, were purified and frozen at −80°C in nutrient broth containing 20% glycerol. Students also returned with the sample a questionnaire in which they identified whether they had stayed or worked in a hospital within the last 2 years.
MSA plates were inoculated with strains frozen at −80°C and cultured overnight at 33–35°C. An aliquot of cells was suspended in 100 μl of water, heated at 100°C for 10 min, then frozen at −20°C until use. 1 μl was used as template in PCR reactions.
Spa typing of the spa gene’s short sequence repeat (SSR) region was performed according to Shopsin et al. , using primers spa1 (5′-AAGACGATCCTTCGGTGAG-3′) and 1517r. Sequencing reactions were performed using GenomeLab™ DTCS Quick Start Kit for Dye Terminator Cycle Sequencing (Beckman Coulter, Inc. Fullerton, CA) and were resolved on a Beckman Coulter CEQ 8800 sequencing machine. spa sequences were determined from both strands of the SSR region. spa repeat sequence type and nomenclature were determined utilizing the Ridom utilities and nomenclature available at http://spaserver.ridom.de/.
SCCmec typing for types I through V was performed according to Boye et al. . Control strains for each of the five SCCmec types (I–V), a gift from the Centers for Disease Control, were used both as positive controls and to act as DNA markers for interpreting the results of the UTPA isolates.
The PCR method of Reischl et al. , was used for the detection of Panton-Valentine leukocidin-positive strains by targeting the lukS-PV gene. The nuc and mecA genes (indicative respectively of S. aureus and resistance to beta-lactam antibiotics) were detected using real-time PCR utilizing the previously reported primers nuc1 and nuc2  and LTmecAF and LTmecAR .
Bacterial identification was performed to the genus level for isolates presented in Table 1 using MicroScan’s POS COMBO 20 dried gram-positive panels, and results interpreted using the Micro Scan autoSCAN-4 system (Dade MicroScan Inc., West Sacramento, CA).
Antibiotic sensitivity was performed on Mueller Hinton agar according to CLSI recommendations for Staphylococcus species (Performance standards for antimicrobial susceptibility testing: Seventeenth Informational supplement. CLSI document M100-S17 (ISBN 1-56238-625-5). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2007). Antibiotics tested were cefoxitin, gentamicin, vancomycin, trimethoprin-sulfamethoxazole, erythromycin, ciprofloxacin, chloramphenicol, and rifampicin.
Fifty seven bacteria were isolated from 375 nasal swabs. Characterization of a sub-set of these isolates is presented in Table 1, all of which were shown to be Staphylococcus species. Seven of the 57 bacteria tested positive for both the nuc and spa genes, and were considered to be S. aureus. There was complete agreement between the nuc/spa tests and no cases existed where an isolate tested positive for nuc and not spa, or visa versa. Of the seven S. aureus isolates, six of them were found to posses the mecA gene and were considered to be MRSA.
Control strains for SCCmec types I–V all typed correctly and confirmed the proper functioning of the test, which identified a bacterial landscape surprisingly rich in different SCCmec types. Five of the six MRSA strains were shown to be of type IV, characteristic of community-associated strains. One of the MRSA strains, #28, was untypeable, giving results characteristic of mixed SCCmec types (Table 1 and Fig. 1). SCCmec type IV, and novel-typing patterns indicative of mixed SCCmec types, were common among the non-S. aureus strains examined (Table 2). Eight strains did not produce amplification products during SCCmec typing, of which two were mecA-positive by PCR.
Spa sequence typing identified two strains as USA300, commonly identified with CA-MRSA, along with a USA600 strain. One isolate, #90, was found to contain a novel spa type not present in the spa database at http://spaserver.ridom.de/, (Table 1).
Only one isolate (#38), a USA 300 MRSA strain, was positive for the lukS-PV gene indicating the potential to produce the Panton-Valentine leucocidin cytotoxin.
Antibiotic resistance profiles were determined for both the S. aureus and a subset of non-S. aureus isolates (Table 1).
MRSA, through acquisition of the mobile SCCmec element, acquires genes that confer resistance to a wide range of antibiotics. In areas with higher antibiotic use, such as healthcare facilities, MRSA have been shown to acquire different types of SCCmec elements that confer an increased level of antibiotic resistance, as compared to elements associated with a community setting. Communities along the Texas border with Mexico have access to antibiotics in Mexico without a prescription, thus presumably creating an atypical community environment with higher than normal levels of antibiotic use, and perhaps misuse. This exploratory study sought to provide an initial characterization of MRSA and SCCmec elements in this environment since such data are currently lacking. Samples were collected from a community environment among presumably healthy individuals where the predominant SCCmec type is type IV, . It was therefore not surprising that five of the six MRSA isolated in this study were SCCmec type IV. In fact, even among non-S. aureus staphylococcal strains, SCCmec type IV was the most common of the five SCCmec types examined (Table 2). One of the predominant type IV strains of MRSA in the U.S. is the USA300 strain, , and two of the six strains did possess the spa type associated with this strain. However only one of these two strains contained the lukS-PV gene, suggesting that the two USA300 strains might not be closely related.
Only the two USA300 strains gave the same spa type, suggesting a relatively high level of diversity among the MRSA isolates and demonstrating the absence of a predominant strain of MRSA among the isolates collected in this study. Similarly, only one strain, #90, gave a spa type not present in the database of spa types used in this study, suggesting that the study area was not enriched with novel strains of MRSA. However the study was limited in scope and it is possible that an examination of a larger number of MRSA strains could reveal a predominant strain(s) for the region.
Characteristic of bacteria with SCCmec type IV elements associated with CA-MRSA strains, most of the MRSA strains (four out of six) were resistant primarily to beta-lactam antibiotics (Table 1). However two of these strains were found to contain a mecA gene but were not resistant to cefoxitin. A possible explanation for this apparent contradiction is that the strains may express heterogeneus resistance to beta-lactams, which is known to occur with some MRSA strains . Among the non-beta-lactam antibiotics tested, resistance to erythromycin was relatively more prevalent, the significance of which is not clear, but might reflect its use in the region. It is also interesting that, like HA-MRSA, the non-S. aureus staphylococcal strains examined generally showed resistance to more types and numbers of antibiotics than the CA-MRSA strains, including resistance to three or four different classes of antibiotics (Table 1). However, although intriguing, it is not yet known if the increased resistance observed among the non-S. aureus strains is located on the SCCmec element. If the increased resistance to antibiotics observed in the non-S. aureus staphylococcal strains is found to be located on the SCCmec element, these bacteria may act as a source of antibiotic resistance for S. aureus via the SCCmec element, and would have to be viewed as an integral part of the problem MRSA represents to the healthcare system. It is noteworthy that none of the isolates were resistant to vancomycin, a concern with multi-drug resistance among MRSA.
The high level of genetic diversity observed among the SCCmec elements is particularly interesting (Table 2). Although the S. aureus strains were predominately of SCCmec type IV, the SCCmec elements in non-S. aureus staphylococci were largely untypeable, giving results consistent with a combination of the five established SCCmec types (Fig. 1). There is at least one other report where untypeable SCCmec elements, from Staphylococcus epidermidis in Finland, have been reported to appear as combinations of established SCCmec types . These results suggest that the nasal-derived MRSA are co-existing with other staphylococcal species that contain different, and predominantly atypical, SCCmec elements. This apparent partitioning of SCCmec types between S. aureus and other staphylococcal species co-habiting the anterior nares raises obvious questions regarding the role of these non-S. aureus species in the emergence of SCCmec elements in S. aureus.
Undoubtedly, to eventually understand the dynamics of SCCmec and antibiotic resistance in staphylococcal populations, it will be necessary to understand the dynamics of emerging resistance, including horizontal transfer and fixation of diverse SCCmec elements among the staphylococci . In this regard, the pronounced partitioning of SCCmec types between S. aureus and other Staphylococcus species may represent a useful system of study to answer such questions. In addition, the transitional border environment of south Texas, with its easy access to antibiotics without a prescription, presents itself as a potentially unique and advantageous region through which to perform such studies.
The authors acknowledge the CDC for reference cultures, the Welch Foundation Grant BG-0017 (support for JCG), the HHMI grant #52005209 to UTPA (support for RP), and NIH EARDA grant #5G11HD038438-08.