Longitudinal Case Series of Staphylococcus aureus Colonization and Infection in Two Cohorts of Rural Iowans

Abstract

Objective:

Examine the relationship between colonization with Staphylococcus aureus in the community and symptomatic infection in two cohorts of Iowans.

Design:

Case series within cohort study.

Participants:

Rural Iowans selected from the Keokuk Rural Health Study, the Agricultural Health Study, and the Iowa Voter Registry.

Methods:

Longitudinal study within established cohorts evaluating documented S. aureus infections with samples available for molecular typing.

Results:

We examined this relationship in two cohorts of Iowans with a combined 11 incident cases of S. aureus SSTI, for which samples were available. Seven of the 11 individuals (63.6%) were colonized at baseline, in the nose (3/7, 42.9%), or in both the nose and throat (57.1%). All seven cases had matching sequence types between colonization and infection isolates.

Conclusions:

Staphylococcus aureus causes millions of skin and soft tissue infections yearly. Although colonization with S. aureus is a frequent antecedent to infection, many studies investigating the link between colonization and infection have taken place in a clinical setting, particularly in urban hospitals. Our study has shown similar results in a rural community setting to those previously seen in clinics.

Introduction

S taphylococcus aureus , a pervasive bacterium, resides in almost 30% of the U.S. population^1,2 and is associated with significant morbidity and mortality. Methicillin-resistant S. aureus (MRSA) is an important cause of both healthcare-associated and community-acquired infections with ∼80,000 cases of invasive MRSA infections occurring annually in the United States³ and millions reporting to emergency departments for skin and soft tissue infections (SSTI).⁴ Although S. aureus can colonize many body sites, the most frequent carriage site is the anterior nares of the nose.⁵ The oropharynx and other body sites can also harbor the bacterium,^4,6 yet the prevalence of colonization in these alternative locations has not been investigated thoroughly. While a majority of individuals who are colonized with S. aureus remain asymptomatic,⁷ colonization serves as a risk factor for the development of invasive infections.⁵

The relationship between S. aureus colonization and subsequent infection has been well established among groups, including prisoners,⁸ athletes,⁹ and hospitalized patients,^10,11 who are at high risk for both colonization and infection. However, the majority of studies have focused on closed populations and healthcare facilities. It remains unclear whether S. aureus colonization is associated with an increased risk of infection in community settings, particularly among community populations that have been shown to have unique S. aureus exposure potential—for example, individuals with livestock contact.^12,13 Little is known about the antecedents to community-associated S. aureus infections, as many community members may not seek medical care and/or healthcare providers may not have colonization samples isolated before infection. Moreover, while prior studies have assessed the risk for infection associated with colonization, there are many limitations present. Some have not included molecular typing,^14,15 focused solely on the hospital setting,^10,15–17 or only investigated colonization at the time of an SSTI diagnosis.^8,16,17–20 Therefore, both the concordance rates between colonization and subsequent infection strains, as well as the isolate types, in the community remain uncertain. In the hospital setting, nasal and clinical isolates have exhibited over 70% of shared identity and are primarily healthcare-associated strains^10,11,20; however, some of these studies typed samples at the time of infection, creating temporality concerns.²⁰

The objective of this study is to explore the relationship between S. aureus colonization and subsequent infection in the general population, using longitudinal data from two studies investigating 1,605 rural Iowans, who were tested for colonization at enrollment and followed for 12–18 months to observe infections.

Materials and Methods

Population

Incident and prevalent infections were examined in two cohort studies: one previously described cohort of 1,342 Iowans (1,289 adults, 53 minors), primarily living in rural areas and in contact with livestock¹² (Cohort A) and a second cohort which enrolled 263 individuals (177 adults, 86 minors) in 95 Iowa families (Cohort B).²¹ Nose and throat samples were taken from adults in both cohorts for isolation of S. aureus at enrollment; minors were swabbed in the nose only. Although no further colonization samples were taken from Cohort A, participants filled out monthly questionnaires (n = 18, enrollment and 17 monthly follow-up questionnaires) asking about possible and/or physician-confirmed S. aureus infections. Cohort B submitted weekly self-collected nose and throat swabs, and also completed weekly questionnaires (n = 52, enrollment and 51 weekly follow-up questionnaires) regarding potential or physician-confirmed S. aureus infections. Both cohorts were encouraged to send samples of potential infections to study investigators, and were provided with instructions and packaging to do so. Samples were cultured and typed as previously described.¹² All participants signed informed consent documents approved by the University of Iowa and the National Institutes of Health Institutional Review Boards (IRB #201101780).

Case definition

All laboratory-confirmed infections collected during the studies from enrollment to the end of the follow-up period were included.

Molecular and phenotypic testing

PVL gene presence was determined as previously described, using the primer pair lukS-PV and lukF-PV.²² The mecA gene was amplified in a multiplex reaction that also amplified the bacterial 16S rRNA and nuc genes for quality control.²³ scn PCR was performed on all isolates using primers described previously.²⁴ The spa repeat region was amplified using primers spa 1113f and spa 1514r, as previously described.²⁵ PCR products were purified using the Qiagen PCR Purification Kit (Qiagen) and sequenced with the same primers used for amplification. Sequences were analyzed using the Ridom StaphType software package (Ridom GmbH) to obtain spa types, which were grouped into spa cluster complexes using the Based Upon Repeat Pattern clustering algorithm.^26,27 Multilocus sequence typing (MLST) was carried out as previously described for select isolates.²⁸

Using the Clinical and Laboratory Standards Institute²⁹ methodology, isolates were tested for susceptibility to a panel of antibiotics, including oxacillin, tetracycline, erythromycin, clindamycin, trimethoprim–sulfamethoxazole, gentamicin, levofloxacin, imipenem, linezolid, daptomycin, vancomycin, quinupristin/dalfopristin, rifampin, inducible clindamycin, and high-level mupirocin.

Statistical analyses

Chi-square analyses were conducted to assess the difference in baseline point prevalence of colonization among all cases and Cohort A (excluding cases). Additionally, Fisher's Exact Test assessed the difference in nasal colonization between cases and cohort A, as well as the difference in colonization at both anatomical sites between cases and cohort A. Similar comparisons were unable to be made within Cohort B due to limitations in the study design. Analyses were conducted with a significance level of α = 0.05.

Results

Infections

Ten individuals had confirmed and typed incident S. aureus infections from Cohort A. Putative infection swabs were received from 22 participants; swabs from 12 participants were culture-negative for S. aureus.

One individual from Cohort B had a confirmed and typed incident S. aureus infection. Putative infection swabs were received from four individuals; three were culture-negative for S. aureus.

Of 11 individuals with laboratory isolation and typing of an S. aureus infection, 7/11 (63.6%) of individuals were colonized at baseline, in the nose (3/7, 42.9%) or in both the nose and throat (4/7, 57.1%). No cases were colonized only in the throat.

Among all cases, the point prevalence of S. aureus colonization (7/11, 63.5%) was higher than among Cohort A participants at baseline (Cohort A: 351/1,332, 26.4%, p = 0.005). Although nasal-only S. aureus colonization in all our cases was lower than Cohort A as a whole (3/7, 42.9% among cases versus 215/351, 61.3% in Cohort A), this was not significant (p = 0.44). Baseline prevalence of S. aureus colonization at both anatomical sites was also not significantly higher among cases than in Cohort A overall (4/7, 57.1% versus 82/351, 23.4%, p = 0.06).”

Molecular and phenotypic typing of colonization and infection isolates

All colonized individuals were infected with the same molecular type of S. aureus by MLST, although some variation was seen phenotypically (cases 2 and 5) and in spa type (case 6). Case 11 from cohort B was sampled weekly, and while t1921 was the most common spa type identified, four other spa types were found during her one-year follow-up (Table 1).

Table 1.

Characteristics of Cases and Colonization/Infection Samples

ID	Sex, age	Colonization or infection	Date	Antibiotic resistance	mecA	spa, MLST	PVL	scn	Animal exposures	Description of infection	Treatment
1	M, 78	Colonization, nose	Jun. 11	OXA, ERY, LVX	+	t008, ST8	+	−	None
		Infection	Jun. 11	OXA, ERY, LVX	+	t008, ST8	+	−	None	Dry, flaky skin	—
		Infection	Nov. 11	OXA, ERY, LVX	+	t008, ST8	+	−	None	Rash, cellulitis, draining pus, impetigo	Linezolid
2	M, 61	Colonization, nose and throat	Jul. 11	TET, SXT, LVX	−	t571, ST398	−	−	SW, C
2	M, 61	Infection	Jul. 11	TET, SXT, LVX	−	t571, ST398	−	−	SW, C	Cellulitis	Ciprofloxacin
		Infection	Nov. 11	TET, SXT, LVX	−	t571, ST398	−	−	SW, C	Cellulitis, draining pus, headache, ill feeling	Ciprofloxacin, Neosporin, warm compress, incision, draining
		Infection	Aug. 12	TET, SXT, LVX	−	t571, ST398	−	−	SW, C	—	Mupirocin, SXT, Ciprofloxacin, warm compress, drainage
		Infection	Aug. 12	OXA, TET, SXT, LVX	−	t571, ST398	−	−	SW, C	—	Mupirocin, SXT, Ciprofloxacin, warm compress, drainage
3	M, 54	Colonization, nose and throat	May 11	None	−	t179, ST5	−	+	None
3	M, 54	Infection	Jul. 11	None	−	t179, ST5	−	+	None	—	—
4	M, 79	Not colonized	Jun. 11
4	M, 79	Infection	Aug. 11	OXA, ERY	+	t008, ST8	+	+	None	—	—
5	M, not provided	Colonization, nose and throat	Jul. 11	ERY	−	t338, ST30
5	M, not provided	Infection	Aug. 11	ERY, iCLI	−	t338, ST30	−	−	None	—	—
6	M, 14	Colonization, nose	Jun. 11	TET	−	t1456, ST398	−	−	C, CH, SH
6	M, 14	Infection	Jul. 12	TET	−	t1250, ST398	−	−	C, CH, SH	—	Antifungal cream
7	M, 68	Not colonized	Jul. 11
7	M, 68	Infection	Jul. 12	TET	−	t3496, ST398	−	−	SW, C, CH, D, G	—	Incision and draining Neosporin, warm compress
8	M, 78	Colonization, nose	Aug. 11	ERY	−	t008, ST8	+	+	None
8	M, 78	Infection	Sep. 12	ERY	−	t008, ST8	+	+	None	—	—
9	M, 54	Not colonized	Aug. 11
9	M, 54	Infection	Oct. 12	NONE	−	t269, ST121	−	+	None	—	—
10	M, 77	Not colonized	Aug. 11
10	M, 77	Infection	Nov. 12	ERY	−	t084, ST15	−	+	SW, C	Folliculitis	Clindamycin, mupirocin
11	F, 26	Colonized, nose and throat	^*	ERY, iCLI	−	t1921^* ST30	−	+	D, H
11		Infection	May 12	ERY, iCLI	−	t1921, ST30	−	+	D, H		Azithromycin

Participant was sampled weekly for 1 year and was a persistent carrier. Throat exclusively t1921; nose most commonly t1921, but also t399 (2 weeks); t084 (1 week); t342 (1 week); t002 (1 week). Case 11 was from Cohort B; all other cases were from Cohort A.

C, cattle; CH, chicken; D, dog; ERY, erythromycin; G, goat; H, horse; iCLI, inducible clindamycin; LVX, levofloxacin; MLST, multilocus sequence typing; OXA, oxacillin; TET, tetracycline; SH, sheep; SXT, trimethoprim–sulfamethoxazole; SW, swine.

Among the 11 individuals, 6 different MLST types and 10 different spa types were identified. Three individuals had ST398 infections, the prototypical “livestock-associated” strain of S. aureus. All three of these individuals reported contact with cattle; two additionally had contact with swine and two with chickens. All ST398 infections were methicillin susceptible and negative for the methicillin resistance gene, mecA. All ST398 infection isolates were negative for scn and tetracycline resistant. One case (Case 2) had a prevalent ST398 infection at enrollment and was colonized in the nose and throat with the same strain; two documented reinfections occurred 4 and 13 months postenrollment. Case 6 was also colonized with ST398 13 months before his infection with ST398 of a different spa type. The third case with an ST398 infection, Case 7, was not colonized at enrollment.

Three individuals, all lacking livestock exposure, were infected with strains carrying PVL, a putative virulence factor. All PVL-positive isolates were ST8. Case 1 had a prevalent infection at enrollment and a second confirmed ST8 infection 5 months postenrollment. Case 8 was colonized at enrollment and had a confirmed ST8 infection 11 months postenrollment. Colonization was not detected at enrollment in Case 4, but he had a confirmed ST8 infection 2 months postenrollment.

Two infected individuals were found to have different spa types of ST30. Both were colonized at enrollment. Case 5 was enrolled in July with spa type t338 and reported an infection one month later with the same type. Case 11 was colonized in February with spa type t1921 and reported an infection 3 months later with the same type.

The remaining cases showed three different sequence types: ST5 (Case 3), ST121 (Case 9), and ST15 (Case 10). Of these, only Case 3 was colonized at enrollment, and reported infection one month later. Case 9 reported infection 14 months postenrollment, and Case 10 15 months postenrollment.

Discussion

While a number of studies have suggested that colonization with S. aureus leads to a higher risk of subsequent infection,^8,9 relatively few longitudinal research studies have been conducted on this topic in a community setting. There are a number of reasons for this: a large cohort of individuals needs to be recruited and followed to observe relatively few infections; individuals may be lost to follow-up over time; and the ease of diagnosis and sample specimen collection within a community setting presents significant challenges not present in a clinical or hospital setting.

Compared with Cohort A as a whole, our infected cases were significantly more likely to be colonized at baseline. This may be indicative of higher pathogen loads or persistent carriage⁵ in the population with incident infections than in the cohort overall. Like previously reported research from patients in hospitals and clinics, individuals who developed symptomatic infections had isolates that closely matched their colonization strains, when present,^10,11,20 suggesting infection stemmed from the host's own colonization strains; colonization ensued from exposure to the infection strain; or that the host was both colonized and infected from a recurrent environmental exposure (including, potentially, other animal species).

For the individual who was divergent in spa type from enrollment to infection (case 6), there are a number of potential scenarios, and our study design does not allow us to determine which is most likely. As we only typed a single isolate per individual per time point, it is possible that one or both time points could have contained a mixed population of both strains (t1456 and t1250, both ST398). As these spa types are closely related, a loss of two repeats by t1250 would result in identification of t1456 (spa repeats 08-16-02-25-02-25 for t1250 versus 08-16-02-25 for t1456). Because this individual had contact with a number of animals (swine, cattle, chickens, dogs, and goats), he may have also been exposed to multiple types of S. aureus from various animal species.

Our confirmed infections showed the same number of ST398 and ST8 infections. ST8 includes USA300, a common community-associated strain of S. aureus, which is generally PVL positive and is a frequent cause of SSTI as well as invasive infections.³⁰ Participants with both sequence types had very similar dynamics of colonization and infection. One individual in each group presented at enrollment with a prevalent infection and also had later incident infections (Cases 1 and 2), another individual in each group was colonized at baseline with an incident infection 13 months later (Case 6, ST398, and Case 8, ST8), and one individual with each sequence type was negative for colonization at enrollment but later had infections due to these sequence types (Cases 4 and 7). Although confirmed ST398 infection isolates remain relatively rare in the U.S., this is further evidence that, when present, livestock-associated ST398 strains have a similar ability to cause infection as more common strains (such as ST8 and ST5, Case 3).

A strength of our study was the large number of individuals investigated (1,605 between the two cohorts) and the length of follow-up (12–18 months). Additionally, we examined colonization and infection with any S. aureus, including isolates that were susceptible to methicillin rather than solely focusing on MRSA. Indeed, in our infection isolates, only two were MRSA.

Our study has a number of limitations. In Cohort A, we have colonization data only at enrollment. This may have missed individuals who were intermediate carriers, or who were persistent carriers but for some reason tested negative in their enrollment sample.

We also were unable to perform molecular typing on most reported S. aureus infections. Twenty additional individuals in Cohort A and one additional individual in Cohort B reported diagnosis of a physician-confirmed S. aureus infection, but did not include a culture swab or bacterial isolate for molecular testing. As such, we cannot match infection isolates to colonization isolates for these individuals. For other putative infection samples, recovery of S. aureus even from a sample swab would be unlikely (for example, cellulitis). As such, our reported incident infections represent a minimum for these cohorts.

Furthermore, our studies were powered to detect differences in colonization among individuals with and without livestock exposure, rather than to detect infections. As such, these may be underpowered for this aspect, and larger studies are necessary to further investigate this phenomenon in the community.

Finally, our results may not be generalizable to all populations. Our cohorts consisted largely of rural Iowans, including those with livestock exposure. Of the 11 cases, 5 self-reported exposure to animals. These were primarily farm animals, which would be an uncommon exposure in the general population. Three of these livestock-exposed cases also demonstrated infection with ST398, the prototypical livestock-associated S. aureus strain, a much higher percentage than would be expected in the general population. Nevertheless, they support the conclusion that S. aureus infection in the community is largely a result of strains carried by the host or encountered in their environment, similar to that seen in hospitalized individuals.

Acknowledgments

This work was supported in part by the Agency for Healthcare Research and Quality (R18 HS019966) and by AFRI food safety grant (#2011-67005-30337) from the USDA National Institute of Food and Agriculture (TCS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Gorwitz

R.J.

, Kruszon-Moran

, McAllister

S.K.

, McQuillan

, McDougal

L.K.

, Fosheim

G.E.

, Jensen

B.J.

, Killgore

, Tenover

F.C.

, and Kuehnert

M.J.

. 2008. Changes in the prevalence of nasal colonization with Staphylococcus aureus in the United States, 2001–2004. J. Infect. Dis., 197:1226–1234.

Kuehnert

M.J.

, Kruszon-Moran

, Hill

H.A.

, McQuillan

, McAllister

S.K.

, Fosheim

, McDougal

L.K.

, Chaitram

, Jensen

, and Fridkin

S.K.

. 2006. Prevalence of Staphylococcus aureus nasal colonization in the United States, 2001–2002. J. Infect. Dis., 193:172–179.

Dantes

, Mu

, Belflower

, Aragon

, Dumyati

, Harrison

L.H.

, Lessa

F.C.

, Lynfield

, Nadle

, and Petit

. 2013. National burden of invasive methicillin-resistant Staphylococcus aureus infections, United States, 2011. JAMA Intern. Med., 173:1970–1978.

Pallin

D.J.

, Egan

D.J.

, Pelletier

A.J.

, Espinola

J.A.

, Hooper

D.C.

, and Camargo

C.A.

. 2008. Increased US emergency department visits for skin and soft tissue infections, and changes in antibiotic choices, during the emergence of community-associated methicillin-resistant Staphylococcus aureus. Ann. Emerg. Med., 51:291–298.

Wertheim

H.F.

, Melles

D.C.

, Vos

M.C.

, van Leeuwen

, van Belkum

, Verbrugh

H.A.

, and Nouwen

J.L.

. 2005. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect. Dis., 5:751–762.

Faden

, Lesse

A.J.

, Trask

, Hill

J.A.

, Hess

D.J.

, Dryja

, and Lee

Y.H.

. 2010. Importance of colonization site in the current epidemic of staphylococcal skin abscesses. Pediatrics, 125:e618–e624.

Graham

P.L.

, Lin

S.X.

, and Larson

E.L.

. 2006. A US population-based survey of Staphylococcus aureus colonization. Ann. Intern. Med., 144:318–325.

Miko

B.A.

, Befus

, Herzig

C.T.

, Mukherjee

D.V.

, Apa

Z.L.

, Bai

R.Y.

, Tanner

J.P.

, Gage

, Genovese

, and Koenigsmann

C.J.

. 2015. Epidemiological and biological determinants of Staphylococcus aureus clinical infection in New York State maximum security prisons. Clin. Infect. Dis., 61:203–210.

Jiménez-Truque

, Saye

E.J.

, Soper

, Saville

B.R.

, Thomsen

, Edwards

K.M.

, and Creech

C.B.

. 2016. Longitudinal assessment of colonization with Staphylococcus aureus in healthy collegiate athletes. J. Pediatr. Infect. Dis. Soc., 5:105–113.

10.

Stenehjem

, Crispell

E.K.

, Rimland

, Farley

M.M.

, and Satola

S.W.

. 2015. Longitudinal evaluation of clinical and colonization Methicillin-Resistant Staphylococcus aureus isolates among veterans. Infect. Control Hosp. Epidemiol., 36:587–589.

11.

Kao

K.C.

, Chen

C.B.

, Hu

H.C.

, Chang

H.C.

, Huang

C.C.

, and Huang

Y.C.

. 2015. Risk factors of methicillin-resistant Staphylococcus aureus infection and correlation with nasal colonization based on molecular genotyping in medical intensive care units: a prospective observational study. Medicine (Baltimore)., 94:e1100.

12.

Wardyn

S.E.

, Forshey

B.M.

, Farina

S.A.

, Kates

A.E.

, Nair

, Quick

M.K.

, Wu

J.Y.

, Hanson

B.M.

, O'Malley

S.M.

, Shows

H.W.

, et al. 2015. Swine farming is a risk factor for infection with and high prevalence of carriage of multidrug-resistant Staphylococcus aureus. Clin. Infect. Dis., 61:59–66.

13.

Nadimpalli

, Stewart

J.R.

, Pierce

, Pisanic

, Love

D.C.

, Hall

, Larsen

, Carroll

K.C.

, Tekle

, Perl

T.M.

, et al. 2016. Livestock-associated, antibiotic-resistant Staphylococcus aureus nasal carriage and recent skin and soft tissue infection among industrial hog operation workers. PLoS One, 11:e0165713.

14.

Azarbal

A.F.

, Harris

, Mitchell

E.L.

, Liem

T.K.

, Landry

G.J.

, McLafferty

R.B.

, Edwards

, and Moneta

G.L.

. 2015. Nasal methicillin-resistant Staphylococcus aureus colonization is associated with increased wound occurrence after major lower extremity amputation. J. Vasc. Surg., 62:401–405.

15.

Cadena

, Thinwa

, Walter

E.A.

, and Frei

C.R.

. 2016. Risk factors for the development of active methicillin-resistant Staphylococcus aureus (MRSA) infection in patients colonized with MRSA at hospital admission. Am. J. Infect. Control, 44:1617–1621.

16.

Kumar

, David

M.Z.

, Boyle-Vavra

, Sieth

, and Daum

R.S.

. 2015. High Staphylococcus aureus colonization prevalence among patients with skin and soft tissue infections and controls in an urban emergency department. J. Clin. Microbiol., 53:810–815.

17.

Albrecht

V.S.

, Limbago

B.M.

, Moran

G.J.

, Krishnadasan

, Gorwitz

R.J.

, McDougal

L.K.

, Talan

D.A.

, and E.M.I.N.S. Group. 2015. Staphylococcus aureus colonization and strain type at various body sites among patients with a closed abscess and uninfected controls at U.S. Emergency Departments. J. Clin. Microbiol., 53:3478–3484.

18.

Ellis

M.W.

, Schlett

C.D.

, Millar

E.V.

, Crawford

K.B.

, Cui

, Lanier

J.B.

, and Tribble

D.R.

. 2014. Prevalence of nasal colonization and strain concordance in patients with community-associated Staphylococcus aureus skin and soft-tissue infections. Infect. Control Hosp. Epidemiol., 35:1251–1256.

19.

Pardos de la Gandara

, Raygoza Garay

J.A.

, Mwangi

, Tobin

J.N.

, Tsang

, Khalida

, D'Orazio

, Kost

R.G.

, Leinberger-Jabari

, Coffran

, et al. 2015. Molecular types of methicillin-resistant Staphylococcus aureus and methicillin-sensitive S. aureus strains causing skin and soft tissue infections and nasal colonization, identified in community health centers in New York City. J. Clin. Microbiol., 53:2648–2658.

20.

Immergluck

L.C.

, Jain

, Ray

S.M.

, Mayberry

, Satola

, Parker

T.C.

, Yuan

, Mohammed

, and Jerris

R.C.

. 2017. Risk of skin and soft tissue infections among children found to be Staphylococcus aureus MRSA USA300 carriers. West. J. Emerg. Med., 18:201–212.

21.

Hanson

B.M.

, Kates

A.E.

, O'Malley

S.M.

, Mills

, Herwaldt

, Torner

J.C.

, Dawson

J.D.

, Farina

, Klostermann

, and Smith

T.C.

. 2017. Staphylococcus aureus colonization and familial transmission over a one year period. BioRxiv. 144782.

22.

Lina

, Piemont

, Godail-Gamot

, Bes

, Peter

M.O.

, Gauduchon

, Vandenesch

, and Etienne

. 1999. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin. Infect. Dis., 29:1128–1132.

23.

Louie

, Goodfellow

, Mathieu

, Glatt

, Louie

, and Simor

A.E.

. 2002. Rapid detection of methicillin-resistant staphylococci from blood culture bottles by using a multiplex PCR assay. J. Clin. Microbiol. 40. 2786–2790.

24.

Price

L.B.

, Stegger

, Hasman

, Aziz

, Larsen

, Andersen

P.S.

, Pearson

, Waters

A.E.

, Foster

J.T.

, Schupp

, et al. 2012. Staphylococcus aureus CC398: host adaptation and emergence of methicillin resistance in livestock. MBio, 3:e00520-12.

25.

Shopsin

, Gomez

, Montgomery

S.O.

, Smith

D.H.

, Waddington

, Dodge

D.E.

, Bost

D.A.

, Riehman

, Naidich

, and Kreiswirth

B.N.

. 1999. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J. Clin. Microbiol., 37:3556–3563.

26.

Mellmann

, Weniger

, Berssenbrugge

, Rothganger

, Sammeth

, Stoye

, and Harmsen

. 2007. Based upon repeat pattern (BURP): an algorithm to characterize the long-term evolution of Staphylococcus aureus populations based on spa polymorphisms. BMC Microbiol., 7:98.

27.

Mellmann

, Weniger

, Berssenbrugge

, Keckevoet

, Friedrich

A.W.

, Harmsen

, and Grundmann

. 2008. Characterization of clonal relatedness among the natural population of Staphylococcus aureus strains by using spa sequence typing and the BURP (based upon repeat patterns) algorithm. J. Clin. Microbiol., 46:2805–2808.

28.

O'Brien

A.M.

, Hanson

B.M.

, Farina

S.A.

, Wu

J.Y.

, Simmering

J.E.

, Wardyn

S.E.

, Forshey

B.M.

, Kulick

M.E.

, Wallinga

D.B.

, and Smith

T.C.

. 2012. MRSA in conventional and alternative retail pork products. PLoS One, 7:e30092.

29.

CLSI. 2009. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Report No.: M07-A7. Clinical and Laboratory Standards Institute, Villanova, PA.

30.

David

M.Z.

, Cadilla

, Boyle-Vavra

, and Daum

R.S.

. 2014. Replacement of HA-MRSA by CA-MRSA infections at an academic medical center in the midwestern United States, 2004–2005 to 2008. PLoS One, 9:e92760.