Abstract
Severe skin lesions caused by Staphylococcus aureus infection are associated with production from bacterial cells of Panton-Valentine leukocidin (PVL), a typical virulence factor of community-acquired methicillin-resistant S. aureus (CA-MRSA), as well as other toxins represented by exfoliative toxins. Through a retrospective study of 26 S. aureus strains isolated from skin lesions of diabetic patients admitted to a hospital in Bangladesh, 2 PVL-gene-positive MRSA-IVa strains and 8 PVL-negative, exfoliative toxin D (ETD) gene (etd)-positive MRSA-IVa strains were isolated. A PVL-positive MRSA-IVa strain had a type I arginine catabolic mobile element (ACME), belonged to ST8/agr-type I/spa-type t121 (a variant of t008), and harbored blaZ, tet(K), msrA, and aph(3′)-IIIa, which are mostly typical characteristics found in USA300, a predominant CA-MRSA clone in the United States. Another PVL-positive MRSA strain, belonging to ST1929 (CC88)/agr-type III/spa-type t3341, was negative for ACME, but possessed blaZ and tet(K). The etd-positive MRSA-IVa strains possessed the epidermal cell differentiation inhibitor B (EDIN-B)–encoding gene (edinB) and belonged to ST1931 (CC80)/agr-type III/spa-type t11023 (a variant of t044), which was genetic trait similar to that of the European CA-MRSA ST80 clone. However, unlike the European ST80 strains, the etd-positive MRSA strains detected in the present study harbored seb, sek, and seq, while they were negative for tet(K), aph(3′)-IIIa, and fusB, showing susceptibility to fusidic acid. These findings suggested that etd-positive ST1931 MRSA strains belong to the same lineage as the European ST80 MRSA clone, evolving from a common ancestral clone via acquisition of a different pathogenicity island. This is the first report of a USA300-like MRSA-IV strain, PVL-positive ST1929 (CC88) MRSA-IV, and European ST80 CA-MRSA-like etd-positive ST1931 (CC80) MRSA-IV strains isolated in Bangladesh.
Introduction
S
The initially identified CA-MRSA strains produce Panton-Valentine leukocidin (PVL), a two-component leukolytic toxin,49,64 which is associated with severe symptoms in a wide spectrum of infections,5,24 including SSTI and necrotizing pneumonia. Recently, it has been recognized that prevalence of CA-MRSA-harboring PVL genes has been increasing in hospitalized patients as well as healthy individuals in the community.27,69 To date, CA-MRSA strains are distributed worldwide and have been shown to be classified into more than 20 distinct genetic lineages, 5 of which are globally predominant: ST1-SCCmec-IV (USA400 clone), ST8-SCCmec-IV (USA300 clone), ST30-SCCmec-IV (South West Pacific clone), ST59-SCCmec-V (Taiwan clone), and ST80-SCCmec-IV (European clone).16,18,41 ST8- and ST30-MRSA-IV are considered as pandemic clones, because of their repeated isolation in every continent.18,45
The USA300 (ST8-MRSA-IV) clone is a predominant CA-MRSA in the United States, Canada, and some Latin American countries. It is acquiring resistance to more antimicrobial agents41,46,61 and becoming a common cause of MRSA infections in healthcare facilities.39,47,51 The strain USA300-0114, which is a variant (PFGE subtype) of the USA300 and represents a dominant clone causing a CA-MRSA epidemic in the United States, 60 belongs to agr-type I and spa-type t008. It is characterized by the presence of type IVa-SCCmec and PVL genes in ΦSA2USA, and the arginine catabolic mobile element (ACME), which is a genetic island integrated into orfX, located adjacent to SCCmec.19,20 The ACME (type I, 30.9 kb) is considered a key factor in the persistence of the USA300 in the community to enhance its fitness and ability to colonize the skin and mucous membranes. 67 Further, ACME contains speG-encoding polyamine N-acetyltransferase, which confers polyamine resistance to bacterial cells, a peculiar trait of the USA300 clone. 31
The European CA-MRSA clone PVL-positive ST80-IV has been isolated increasingly from community-acquired infections in many western European countries, causing nosocomial transmissions,3,9,16,22,27,38,41,50 and has also been found to be highly prevalent among patients in North Africa and the Middle East.8,16,21,41,57,62 In other regions, such as Australia and Malaysia, ST80 (CC80) MRSA was reported at considerably low frequency.2,14,40 The ST80 CA-MRSA clone typically belongs to agr-type III and spa-type t044, and has type IVc-SCCmec and a common toxin profile (etd and edinB genes positive), and shows resistance to β-lactams, kanamycin, tetracycline, and fusidic acid,17,29,32,43 which are traits distinct from those of USA300.
In Bangladesh, prevalence of MRSA has been investigated in a few studies,15,26 and genetic information of circulating MRSA strains is available in only one report in which the PVL gene was detected in ST772 MSSA (Bengal clone) related to the ST1 strain (USA400 clone). 1 However, the prevalence of globally disseminated MRSA clones in Bangladesh has not yet been studied. In the present study, during genetic analysis of the preserved S. aureus isolates from skin infections in diabetic patients in Bangladesh, a PVL-positive USA300-like MRSA and PVL-negative, exfoliative toxin D (ETD)-encoding gene (etd)–positive MRSA strains were identified. Further, the etd-positive MRSA strains were revealed to be related to the ST80 European CA-MRSA clone.
Materials and Methods
Bacterial isolates, initial genetic typing, and major toxin screening
From October 2008 to February 2009, all the diabetic patients with skin infections admitted to a hospital specializing in diabetes in Dhaka, Bangladesh, were examined. S. aureus strains were isolated from 26 diabetic patients with skin infections (specimens: pus, wound swab, diabetic foot ulcer, or burn exudate) (Table 1). These strains were stored in Microbank (Pro-Lab Diagnostics, Richmond Hill, Canada) at −80°C until analysis. For all the isolates, the presence of the staphylococcal 16s rRNA gene, nuc, mecA, PVL gene (lukS-PV/lukF-PV), and ACME-arcA (arginine deiminase gene) was investigated by multiplex PCR assay as described by Zhang et al. 71 SCCmec typing and subtyping, and coagulase gene (coa) typing were also performed by multiplex PCR using previously published primers and conditions.28,36,42 As an initial screening of major virulence factors, three enterotoxins (sea, seb, and sec) and three exfoliative toxins (eta, etb, and etd) were detected by PCR as described previously.7,66
Diabetic foot ulcer.
PVL, Panton-Valentine leukocidin; ACME, arginine catabolic mobile element; SCCmec; staphylococcal cassette chromosome mec.
Antimicrobial susceptibility testing
For selected strains including those with PVL genes or etd, minimum inhibitory concentrations (MICs) against 12 antimicrobial agents (oxacillin, cefoxitin, ampicillin, erythromycin, vancomycin, kanamycin, gentamycin, streptomycin, fusidic acid, fosfomycin, tetracycline, and ciprofloxacin) were measured by the broth microdilution test. Breakpoints defined in the Clinical Laboratory Standards Institute (CLSI 2012) guidelines were employed to distinguish between resistant and susceptible strains. 13
Genetic typing, detection of virulence factors, and drug-resistant genes
For the just-mentioned selected isolates, MLST, spa gene typing, and agr gene typing were performed as described previously.23,54,59 Presence of genes encoding enterotoxins and other toxins, exoenzymes, adhesins, regulatory elements, other proteins related to virulence, and antimicrobial resistance genes were analyzed by multiplex or uniplex PCR using primers described previously4,11,12,33,43,48,55,63 and those designed in the present study (Table 2). For strains positive for ACME by above multiplex PCR, ACME genotypes (I, II, and ΔII) were classified by long-range PCR 1 (LR-PCR 1), in terms of orientation and size of the ACME-arcA locus (LR-PCR 1), the region between ACME-arc and ACME-opp3 (LR-PCR 2) and the region between ACME-opp3 and the copA junction (PCR 3), as previously described.6,34 To know whether the orientation of SCCmec-IV and ACME is identical to that in USA300, LR-PCR was performed with primers ccrA2-rev2 (5′-GTTACAGCTGTGGGAGAAGATGG-3′) and USA300 (0047)-r1 (5′-CATGCCAATGACTTGTTGATCCTC-3′) targeting ccrA2 and SAUSA300_0047, respectively, to amplify the 10,461-bp product.
Primers described by Campbell et al. 11 and Cassat et al. 12 were used for detection of efb and V8 (sspA), and sarT, trap, GntR family protein gene, epiC, isaB, respectively. For detection of fusB, two primer pairs described by Monecke et al. 43 were employed.
Primer previously described by Campbell et al. 11
Regulator protein genes belonging to individual family.
Primer previously described by Cassat et al. 12
Sequence analysis of etd, edinB, ebpS, and coa genes
The ETD-encoding gene (etd) is located adjacent to the epidermal cell differentiation inhibitor B (EDIN-B) gene (edinB) in the chromosome of etd-positive S. aureus strains. 66 Full-length sequences of these genes and their intergenic sequence including an orf2 were determined by PCR with two pairs of primers described in Table 2, and direct sequencing with PCR products. Sequence of the gene encoding elastin-binding protein (ebpS) was determined by PCR and direct sequencing as described previously. 4 For the strains for which the coa genotypes were not determined for I–X by the multiplex PCR, 28 sequences of D1, D2, and the central region of coa were determined as described previously35,65 to assign the coa genotype by sequence homology. Multiple alignment of nucleotide and amino acid sequences determined was performed by the Clustal W program ver. 2.1, which is available on the Web site of DNA Data Bank of Japan (DDBJ) (http://clustalw.ddbj.nig.ac.jp/).
GenBank accession numbers
Sequence of etd and edinB of strain DK-B3 determined in the present study was deposited in the GenBank database under accession number KC609427.
Results
Among the 26 S. aureus strains isolated in the study period, 10 strains were MRSA (mecA-positive) with type IVa-SCCmec belonging to coa-type III or XIa (Table 2). PVL genes were detected in the two coa-type III MRSA strains from diabetic foot ulcers (DK-A8 and DK-A10) and two MSSA strains of coa-type V and VI. The strain DK-A8 was positive for ACME-arcA in type I ACME located downstream from SCCmec-IVa as found in USA300. 20 In contrast, all of the eight coa-type XIa MRSA strains harbored seb and etd.
Genotypes, antimicrobial susceptibility, and presence of drug-resistant genes were analyzed for 12 representative strains: PVL-positive MRSA and MSSA (2 strains each), etd-positive MRSA (4 strains), and PVL-negative MSSA (4 strains including an etd-positive strain) (Table 3). All the MRSA exhibited low levels of resistance to oxacillin (MIC; 16–32 μg/mL), except for a PVL/ACME-positive strain DK-A8 (MIC; 128 μg/mL). Strain DK-A8 belonged to agr-type I, ST8, spa-type t121, and harbored blaZ, tet(K), msrA, and aph(3′)-IIIa showing resistance to ampicillin, erythromycin, kanamycin, fosfomycin, and tetracycline, but lacked mupA and cfr. Another PVL-positive MRSA strain DK-A10 was classified into agr-type III, ST1929 (CC88), spa-type t3341, and had blaZ and tet(K), with resistance to tetracycline. Four etd-positive MRSA strains had identical genetic types, that is, agr-type III, ST1931 (CC80), spa-type t11023, and were susceptible to fusidic acid and most other antimicrobial agents (except for β-lactams), without harboring fusB.
The genes that were negative in all the strains [mupA, cfr, fusB, erm(A), erm(B), erm(C), aac(6′)-Im, aac(6′)-Ie-aph(2″)-Ia, ant(3″)-Ia, ant(4′)-Ia, ant(6)-Ia, ant(9)-Ia, ant(9)-Ib, aph(2″)-Ib, aph(2″)-Ic, and aph(2″)-Id] were not listed.
MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; MIC, minimum inhibitory concentration.
The leukocidin gene lukE-lukD and most of hemolysins-encoding genes were detected in all the strains examined (Table 4). Two PVL-positive MRSA strains had sek and seq (sep in only strain DK-A10), while PVL-positive MSSA strain DK-A3 possessed an enterotoxin gene cluster (seg-sei-sem-sen-seo-seu). All the etd-positive strains were positive for edinB, and had seb, sek, and seq. Nucleotide sequences of etd and edinB, and their intergenic region determined for strains DK-B3 and DK-B5 were 100% identical, and almost identical to the etd-edinB prototype sequence in strain TY11466 (GenBank accession No. AB057421) with nucleotide differences at only four positions (one at each site in etd and edinB and at two sites in the intergenic noncoding region) (data not shown).
Among the adhesin genes examined, map/eap and bbp were detected only in a single strain, PVL-positive ST8 MRSA strain DK-A8 and PVL-positive ST121 MSSA strain DK-A3, respectively (Table 5). Exoenzyme-encoding genes chp and sak were negative in all the etd-positive MRSA strains and a PVL-positive MSSA strain, while splA/splB were detected in all the PVL- or etd-positive MRSA. Although most of the genes encoding the regulatory elements examined were positive in all the strains, the GntR family protein gene was detected in only two strains DK-A8 and DK-A3.
ebpS gene that has 180-bp internal deletion as reported previously. 4
Discussion
Due to enhanced virulence, environmental survival, and transmission properties, the USA300 MRSA clone has spread rapidly within the United States as an important cause of community-acquired and healthcare-associated infections.
46
Although this clone is also disseminated globally as a major international epidemic clone, prevalence outside the Americas has still been low. In the present study, PVL-positive MRSA (two strains) was first detected in Bangladesh, in diabetic foot ulcers of adult patients admitted to a hospital in Dhaka in 2008. One of these strains, DK-A8, showed genotypes identical to the USA300-0114 strain,46,60 and possessed type I ACME and virulence factors found in genomic elements (SaPI5, ΦSA3USA) of the USA300-FPR3757 strain.
19
spa-type t121 (repeat unit profile: 11-19-21-17-34-24-34-22-25) identified for DK-A8 represents a genotype with single-repeat unit deletion of spa-t008 (11-19-
In the present study, it was remarkable that most of the MRSA strains (8 among 10) were etd/edinB positive and PVL negative. Virulence factors etd and edinB have been specifically detected in the European MRSA ST80 clone with SCCmec-IV, associated with agr-type III and predominant spa-type t044.43,51 The etd/edinB-positive MRSA strains analyzed in the present study were classified into ST1931 (MLST profile: 1-3-1-14-11-221-10), a single-locus variant of ST80 (1-3-1-14-11-51-10), and spa-type t11023 (repeat unit profile: 07-23-12-34-34-34-34-34-34-34-34-33-34), which is a variant having multiple internal duplication of a repeat unit in spa-type t044 (07-23-12-34-34-33-34). Similar spa-type variants with repeat-unit duplication (e.g., t376 and t934) have been detected in ST80 (CC80) European CA-MRSA strains.37,53 Therefore, etd/edinB-positive ST1931 MRSA strains detected in the present study are considered to belong to the same lineage as that of European ST80 CA-MRSA (CC80 clonal group). However, some genetic differences (e.g., lack of fusB, and presence of enterotoxin genes) were detected when compared with the European ST80.44,51,58 Accordingly, it can be suggested that the ST1931 MRSA strains might have evolved from an ancestral strain common to that of the European ST80 clone through incorporation of a different pathogenicity island, phage, or plasmid.
Although the SCCmec-IV subtype IVc is predominant in the European ST80 MRSA,29,37 all of the ST1931 MRSA carried SCCmec-IVa. In the study of ST80 CA-MRSA isolated in Denmark, 38 genetic variation (subtype) in the SCCmec-IV appears to be present. As reported for European ST80-MRSA-IV,44,51 protease genes splA and splB were positive in the ST1931 MRSA strains in the present study, while adhesin genes bbp and map were not detected. Therefore, while a high degree of homology was observed for CC80:ST80 CA-MRSA in Europe and the Middle East, 25 it may be possible that outside these regions, genetic variants of the ST80 (CC80) strain are locally predominant as observed for ST1931 MRSA in Bangladesh. A molecular characterization of nasal S. aureus isolates (MSSA) from a remote indigenous African population (Bobongo Pygmies in Gabon) revealed a high prevalence of PVL genes in six STs including ST80, frequently associated with concomitant seb, etd, and edinB. 52 This study suggests that the etd/edinB-positive ST80 S. aureus colonizing in the remote aboriginal population was originated from a same strain as that of ST80 (CC80) European CA-MRSA. Thus, it may be of interest to study the prevalence of etd/edinB-positive CC80 S. aureus strains among healthy populations as well as hospitalized patients in Bangladesh, in order to understand the origin and prevalence of the ST1931 MRSA.
The etd gene is shown to have 40% and 59% identity to eta and etb genes, respectively, and ETD causes blisters in a newborn mouse model. 66 Epidemiological and clinical studies indicated that ETD is associated with cutaneous abscesses and furuncles, while not with bullous impetigo or SSSS (staphylococcus scalded skin syndrome).17,68 However, because etd-positive ST80-MRSA-IV mostly harbors PVL genes, the actual contribution of ETD to the pathogenesis of these MRSA strains has not yet been clearly defined. In the present study, a high prevalence of etd in PVL-negative MRSA isolates was observed. However, to determine the etiologic role of ETD in skin infections, more information from epidemiological and experimental studies is needed.
In conclusion, PVL-positive ST8 MRSA, PVL-positive ST1929 (CC88) MRSA, and PVL-negative/etd-positive ST1931 (CC80) MRSA strains were identified in Bangladesh for the first time. The ST1931 MRSA was considered to belong to the same lineage as that of the European ST80 CA-MRSA, with etd as a distinctive marker for the clonal group CC80. Investigation and analysis of a larger number of the PVL- or etd-positive MRSA strains in Bangladesh might be necessary to verify their importance in public health and to promote infection control against them.
Footnotes
Acknowledgments
This study was supported by a Grant-in-Aid for Scientific Research (No. 23590746) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and the grant provided by the Heiwa Nakajima Foundation.
Disclosure Statement
The authors of this article have no commercial associations that might create a conflict of interest in connection with the submitted article.