Abstract
Prevalence, drug resistance and genetic characteristics were analysed for a total of 128 clinical isolates of staphylococci obtained from a tertiary hospital in Myanmar. The dominant species were S. aureus (39%) and S. haemolyticus (35%), followed by S. epidermidis (6%) and S. saprophyticus (5%). The majority of S. haemolyticus isolates (71.1%) harboured mecA, showing high resistance rates to ampicillin, cephalosporins, erythromycin and levofloxacin, while methicillin-resistant S. aureus (MRSA) was only 8% (four isolates) among S. aureus with type IV SCCmec. Panton-Valentine leukocidin (PVL) genes were detected in 20 isolates of S. aureus (40%), among which only one isolate was MRSA belonging to sequence type (ST) 88/agr-III/coa-IIIa, and the other 19 methicillin-susceptible S. aureus (MSSA) isolates were classified into six STs (ST88, ST121, ST1153, ST1155, ST1930, ST3206). An ST1153 MSSA isolate with PVL was revealed to belong to a novel coa type, XIIIa. ST121 S. aureus was the most common in the PVL-positive MSSA (47%, 9/19), harbouring genes of bone sialoprotein and variant of elastin binding protein as a distinctive feature. Although PVL-positive MSSA was susceptible to most of the antimicrobial agents examined, ST1930 isolates were resistant to erythromycin and levofloxacin. ST59 PVL-negative MRSA and MSSA had more resistance genes than other MRSA and PVL-positive MSSA, showing resistance to more antimicrobial agents. This study indicated higher prevalence of mecA associated with multiple drug resistance in S. haemolyticus than in S. aureus, and dissemination of PVL genes to multiple clones of MSSA, with ST121 being dominant, among hospital isolates in Myanmar.
Keywords: MRSA, Myanmar, Panton-Valentine leukocidin (PVL), ST121, Staphylococcus
Introduction
Staphylococci constitute one of the major normal flora in skin, nasal cavities and mucosal membranes of humans. However, they are known as common causes of various infections in both healthcare and community settings. While approximately 30% of healthy individuals are colonized with Staphylococcus aureus asymptomatically [1], this bacterium causes various infections, including skin and soft tissue infections (SSTI), bactaeremia and pneumonia. Healthcare-associated (HA) methicillin-resistant Staphylococcus aureus (MRSA) has been recognized as a primary cause of nosocomial infections that acquired multiple drug resistance, associated with its global spread since the 1960s [2]. Thereafter, community-acquired (CA) MRSA have also emerged as cause of infections in individuals who have no healthcare-associated risk [3], [4], posing a public health concern worldwide. Coagulase-negative staphylococci (CNS), ubiquitously distributed to humans, have been also increasing as nosocomial pathogens mainly as a result of development of prosthetic devices and invasive medical technologies [5]. Representative species causing infections are S. epidermidis and S. haemolyticus, which often acquire drug resistance, including methicillin resistance via same genetic mechanism as that of MRSA.
Methicillin resistance of staphylococcus is characterized by the presence of a transmissible genome element, staphylococcal cassette chromosome mec (SCCmec), which is inserted in the chromosome of bacterial cell. SCCmec in MRSA has been differentiated into at least 11 genetic types (I–XI) [6], [7], among which types I to III are commonly found in HA-MRSA, while type IV and V were reported to be frequently in CA-MRSA [3]. However, in the present circumstances, CA-MRSA with the dominant SCCmec types have been brought to healthcare settings [8], [9], [10], which makes distinction between HA- and CA-MRSA more difficult in terms of SCCmec type. The initially identified CA-MRSA strains were characterized by production of Panton-Valentine leukocidin (PVL), a two-component leukolytic toxin [11], which is associated with severe symptoms in a wide spectrum of infections [12], [13], including SSTI and necrotizing pneumonia. Prevalence of CA-MRSA harbouring PVL genes has been increasing recently in hospitalized patients as well as healthy individuals in the community [14], [15].
In Myanmar, S. aureus has been reported to be the major pathogen in bloodstream infections and the third most common bacteria in blood cultures from febrile children [16], [17]. However, to our knowledge, there is no epidemiologic study on staphylococci from healthcare settings in Myanmar, and thus information is not available for drug resistance and genetic characteristics on recent clinical isolates of S. aureus, including MRSA, and CNS. Although we previously reported genetic traits of MRSA and methicillin-susceptible S. aureus (MSSA) isolates from hospital, community and food poisoning cases in Myanmar, the epidemiologic features was not determined because of the low numbers of isolates analysed [18]. In the present study, drug resistance and genetic traits, including prevalence of mecA, ACME (arginine catabolic mobile element) and PVL genes, was analysed for clinical isolates of staphylococci in a tertiary-care hospital in Myanmar.
Materials and methods
Bacterial isolates and initial genetic analysis
A total of 128 Staphylococcus strains were collected from patients admitted to North Okkalapa General Hospital, Yangon, Myanmar, between January 2012 and August 2013. The main specimen of the isolates was wound swab of surgical site infections (57%), followed by high vaginal swab (12%), blood (11%), pus (10%) and other specimens (sputum, urine, ear exudate) (10%). A single isolate from an individual patient was subjected to this study. Bacterial isolates grown on agar plates were examined by conventional microbiologic methods, and their species were determined by BBL Crystal Gram-Positive ID Kit (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). Individual bacterial strains were stored in Microbank (Pro-Lab Diagnostics, Richmond Hill, ON, Canada) at −80°C and recovered when they were analysed.
The presence of staphylococcal 16S rRNA, nuc, mecA, PVL gene (lukS-PV/lukF-PV) and ACME-arcA (arginine deiminase gene) were detected for all isolates by multiplex PCR assay as described by Zhang et al. [19]. SCCmec type and ACME type were also determined by multiplex PCR using previously published primers and conditions [20], [21].
Antimicrobial susceptibility testing
For major staphylococcal species, minimum inhibitory concentrations against 18 antimicrobial agents based on the broth microdilution test were measured by using Dry Plate ‘Eiken’ DP32 (Eiken Chemical, Tokyo, Japan) for Gram-positive cocci. Breakpoints defined in the Clinical Laboratory Standards Institute (CLSI) guidelines were used to distinguish between resistant and susceptible strains for most of the drugs examined [22].
Genetic typing, detection of virulence factors and drug resistance genes of S. aureus
The staphylocoagulase genotype (coa type) of S. aureus isolates was determined by multiplex PCR using previously published primers and conditions [23]. For the strains for which the coa types were not determined for I–X by the multiplex PCR, sequences of D1, D2 and the central region of coa were determined as described previously [24], [25] to assign the coa genotype by sequence homology. Sequence identity to the known coa types was analysed by Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov/Blast.cgi). For selected isolates, sequence type (ST) was determined according to the scheme of multilocus sequence typing (MLST) [26], and agr group classification and protein A gene (spa) typing were performed as described previously [27], [28].
The presence of genes encoding enterotoxins and other toxins, adhesins, other proteins related to virulence and antimicrobial resistance genes were analysed by multiplex or uniplex PCR using primers described previously [18]. Partial sequence of the gene encoding elastin-binding protein (ebpS) was determined by PCR and direct sequencing as described previously [18]. Multiple alignment of nucleotide and amino acid sequences determined was performed by the CLUSTAL W 2.1 program (DNA Data Bank of Japan (DDBJ), http://clustalw.ddbj.nig.ac.jp/).
Full-length staphylocoagulase gene (coa) sequence of strain MMR-v determined in the present study was deposited in the GenBank database under accession number KT599478.
Results
Among the 128 staphylococcal isolates obtained in the study period, the dominant species identified were S. aureus (n = 50, 39%) and S. haemolyticus (n = 45, 35%), followed by S. epidermidis (n = 8, 6%) and S. saprophyticus (n = 7, 5%). S. aureus and S. haemolyticus were isolated from wound swabs at high rates (65% and 62%, respectively), while S. haemolyticus was the main species among isolates from blood culture (43%, 6/14). The majority of S. haemolyticus (71%, 32/45) and S. epidermidis (75%, 6/8) possessed mecA, while the detection rate of MRSA was only 8% (4/50), and all the four MRSA had type IV SCCmec (Table 1). Although the SCCmec of some S. haemolyticus and S. epidermidis isolates was assigned to types IV and V, most of the isolates (71%, 27/38) were untypable. ACME-arcA was detected in two and one isolates of S. haemolyticus and S. epidermidis, respectively, and their ACME was classified into type II. PVL genes were detected in 20 S. aureus isolates (40%), among which only one isolate was MRSA. PVL-positive S. aureus were mostly isolated from pus or wound swabs (Table 2).
Table 1.
Frequencies of isolates with PVL genes, ACME and mecA (SCCmec type) among different Staphylococcus species
| Staphylococcus species | mecA | No. of isolates | PVL genes (+) | ACME-arcA (+) (ACME type) | SCCmec type |
|||
|---|---|---|---|---|---|---|---|---|
| II | IV | V | NT | |||||
| S. aureus (n = 50) | + | 4 | 1 | 0 | 4 | |||
| − | 46 | 19 | 0 | |||||
| S. haemolyticus (n = 45) | + | 32 | 0 | 1 (ACMEII) | 2 | 5 | 25 | |
| − | 13 | 0 | 1 (ACMEII) | |||||
| S. epidermidis (n = 8) | + | 6 | 0 | 0 | 3 | 1 | 2 | |
| − | 2 | 0 | 1 (ACMEII) | |||||
| S. saprophyticus (n = 7) | + | 1 | 0 | 0 | 1 | |||
| − | 6 | 0 | 0 | |||||
| Other (n = 18) | + | 3a | 0 | 0 | 1b | 2 | ||
| − | 15c | 0 | 0 | |||||
PVL, Panton-Valentine leukocidin; NT, nontypeable.
One isolate each of S. hominis, S. sciuri and S. vitulinus.
S. sciuri.
S. auricularis (1), S. capitis (1), S. cohnii (1), S. hominis (1), S. kloosii (3), S. sciuri (1), S. vitulinus (1), S. warneri (3), S. xylosus (3).
Table 2.
Genotype (ST) of PVL-positive Staphylococcus aureus isolates
| mecA | coa type | ST | CCa | No. of isolates | Specimen (n) |
|---|---|---|---|---|---|
| + | IIIa | ST88 | CC88 | 1 | Wound (1) |
| − | IIIa | ST88 | CC88 | 2 | Blood (1), wound (1) |
| − | Va | ST121 | CC121 | 9 | Pus (2), wound (7) |
| − | VIa | ST1930 | CC96 | 3 | Pus (1), wound (2) |
| − | VIa | ST3206 | CC1 | 2 | Pus (1), wound (1) |
| − | VIIa | ST1155 | CC101 | 2 | Pus (1), high vaginal swab (1) |
| − | XIIIa | ST1153 | CC1153 | 1 | Wound (1) |
CC, clonal complex; PVL, Panton-Valentine leukocidin; ST, sequence type.
CC of ST.
While the four MRSA isolates showed resistance to oxacillin and ampicillin, they were mostly susceptible to all other antimicrobial agents, except for gentamicin and erythromycin (Table 3). MSSA isolates were susceptible to most antimicrobial agents while showing low resistance rates to ampicillin, erythromycin, gentamicin and sulfamethoxazole/trimethoprim. In contrast, mecA-positive S. haemolyticus showed high resistance rates to ampicillin, cephalosporins, erythromycin and levofloxacin. Similar to MRSA, mecA-positive S. epidermidis were susceptible to most of the antimicrobial agents except for oxacillin. None of the staphylococcal isolates was resistant to vancomycin, linezolid and fosfomycin.
Table 3.
Resistance rates of Staphylococcus species against antimicrobial agents
| Antimicrobial agenta | Resistant isolates, n (%) |
|||||
|---|---|---|---|---|---|---|
|
S. aureus |
S. haemolyticus |
S. epidermidis |
||||
| mecA(+) (n = 4) | mecA(−) (n = 46) | mecA(+) (n = 32) | mecA(−) (n = 13) | mecA(+) (n = 6) | mecA(−) (n = 2) | |
| OXA | 4 (100) | 0 (0) | 29 (90.6) | 4 (30.8) | 6 (100) | 1 (50) |
| FOX | 1 (25) | 0 (0) | 28 (87.5) | 2 (15.4) | 0 (0) | 2 (100) |
| AMP | 4 (100) | 15 (32.6) | 28 (87.5) | 1 (7.7) | 1 (16.7) | 0 (0) |
| CFZ | 0 (0) | 0 (0) | 23 (71.9) | 1 (7.7) | 0 (0) | 0 (0) |
| CMZ | 0 (0) | 0 (0) | 17 (53.1) | 1 (7.7) | 0 (0) | 1 (50) |
| FMX | 0 (0) | 0 (0) | 7 (21.9) | 2 (15.4) | 0 (0) | 1 (50) |
| IPM | 0 (0) | 0 (0) | 14 (43.8) | 0 (0) | 0 (0) | 0 (0) |
| GEN | 2 (50) | 6 (13) | 25 (78.1) | 0 (0) | 1 (16.7) | 1 (50) |
| ABK | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 1 (16.7) | 1 (50) |
| MIN | 1 (25) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| ERY | 2 (50) | 7 (15.2) | 30 (93.8) | 6 (46.2) | 0 (0) | 2 (100) |
| CLI | 1 (25) | 6 (13) | 6 (18.8) | 3 (23.1) | 1 (16.7) | 1 (50) |
| VAN | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| TEC | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| LZD | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| FOF | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
| LVX | 1 (25) | 3 (6.5) | 28 (87.5) | 1 (7.7) | 3 (50) | 1 (50) |
| STX | 1 (25) | 7 (15.2) | 20 (62.5) | 1 (7.7) | 3 (50) | 1 (50) |
ABK, arbekacin; AMP, ampicillin; CFZ, cefazolin; CLI, clindamycin; CMZ, cefmetazole; ERY, erythromycin; FMX, flomoxef; FOF, fosfomycin; FOX, cefoxitin; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; LZD, linezolid; MIN, minocycline; OXA, oxacillin; SXT, sulfamethoxazole/trimethoprim; TEC, teicoplanin; VAN, vancomycin.
Resistance to individual antimicrobial agents was judged according to Clinical Laboratory Standards Institute (CLSI) guidelines. For antimicrobial agents whose resistance is not defined by CLSI guidelines, European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (Staphylococcus spp., FOF, >32 μg/mL) and the following definitions (minimum inhibitory concentration) were used to determine resistance for S. aureus and S. haemolyticus: ABK, >4 μg/mL.
Among the 50 S. aureus isolates, ten staphylocoagulase (coa) genotypes were identified by multiplex PCR or sequencing, and the most common type was Va (n = 19), followed by VIIa (n = 8), VIa (n = 6), and IIIa and VIIb (n = 4) (Table 4). Full-length coa was determined for an MSSA strain MMR-v of which the coa type was untypable by the PCR assay. Sequence identity of coa-D1 region and -D2 plus central regions of MMR-v to the known 12 coa types were 64.7–70.3% and 69.7–89.2%, respectively (Supplementary Table S1). According to the criteria to determine coa type (subtype) proposed by Watanabe et al. [25], i.e. >90% identity of the D1 region (coa type) and >90% identity of the D1 and central region (coa subtype), the staphylocoagulase gene of MMR-v was considered not to be classified into the known 12 coa types. Therefore, a new coa type, XIIIa, was assigned to this strain. While MRSA belonged to three coa types (IIIa, IVb, VIIb), PVL-positive isolates were assigned to five coa types (IIIa, Va, VIa, VIIa, XIIIa), with Va being dominant, followed by VIa.
Table 4.
Frequencies of PVL and mecA genes among different coa genotype of Staphylococcus aureus isolates
| coa type | No. of isolates | PVL(+) | mecA(+) |
|---|---|---|---|
| IIa | 2 | ||
| IIIa | 4 | 3 | 1 |
| IVb | 2 | 1 | |
| Va | 19 | 9 | |
| Vb | 1 | ||
| VIa | 6 | 5 | |
| VIIa | 8 | 2 | |
| VIIb | 4 | 2 | |
| Xa | 3 | ||
| XIIIa | 1 | 1 | |
| Total | 50 | 20 | 4 |
PVL, Panton-Valentine leukocidin.
MLST was performed for 27 isolates, i.e. 20 PVL-positive and 7 PVL-negative S. aureus isolates, resulting in identification of 11 STs (Table 2, Table 5). ST3206 (CC1) of two PVL-positive MSSA isolates and ST3075 of a PVL-negative MSSA isolate were newly identified in the present study. PVL-positive isolates were differentiated into six STs (ST88, ST121, ST1153, ST1155, ST1930, ST3206), among which ST121 was dominant (nine isolates, 45% of PVL-positive S. aureus) and found in only MSSA, and other STs were identified in one to three isolates. A PVL-positive MRSA, strain MMR-42A, belonged to ST88, coa type IIIa, agr type III, and spa type t729. The other three MRSA isolates were classified into ST6 and ST59 (Table 5). ST88 was also identified in PVL-positive MSSA from blood and wound, which exhibited different patterns of toxin/virulence factors and drug resistance from those of a PVL-positive ST88 MRSA. ST121 PVL-positive MSSA isolates belonged to coa type Va and agr type IV, and harboured five to six enterotoxin genes, the bone sialoprotein gene (bbp), and a variant of the elastin binding protein gene (ebpS-v) with an internal 180 bp deletion as described previously [18]. The ST59 S. aureus, both MRSA (two strains) and MSSA (one strain) without PVL had more resistance genes (ermB, aac(6′)-Ie-aph(2″)-Ia) than other MRSA and PVL-positive MSSA, showing resistance to more antimicrobial agents.
Table 5.
Genotypes, virulence factors and drug resistance in 15 MSSA and MRSA strains
| mecA/PVL genes | Strain ID | Age/Sex | Specimen | Genotype |
Leukocidins, haemolysinsa | Enterotoxinsb | Adhesins and othera | Drug resistance genec | Antimicrobial resistance patternd | |||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| SCC mec | coa | agr | ST (CC) | |||||||||
| PVL | MMR-20A | 34/M | Blood | IIIa | III | ST88 (CC88) | lukE-lukD, hla, hlg2 | seq | sdrC, sdrD, sdrE, fib, clfB, ebpS | blaZ | AMP | |
| PVL | MMR-k | 36/F | Wound | Va | IV | ST121 (CC121) | lukE-lukD, hla, hlg2 | seb, sei, sem, sen, seo | fib, clfB, cna, bbp, ebpS-v | blaZ | AMP | |
| PVL | MMR-6A | 30/M | Pus | Va | IV | ST121 (CC121) | lukE-lukD, hla, hlg2 | seb, seg, sei, sem, sen, seo | fib, clfB, cna, bbp, ebpS-v | blaZ | AMP | |
| PVL | MMR-z0 | 38/M | Wound | VIa | III | ST1930 (CC96) | lukE-lukD, hla, hlg2 | sea, sec, sei, sel | sdrC, sdrD, sdrE, fib, clfB, ebpS, cna | blaZ, ermC | AMP, ERY, LVX | |
| PVL | MMR-46A | 49/M | Wound | VIa | III | ST1930 (CC96) | lukE-lukD, hla, hlg2 | sea, sec, sei, sel | sdrC, sdrD, sdrE, fib, clfB, ebpS | blaZ, ermC | AMP, ERY, LVX | |
| PVL | MMR-30B | 30/F | Pus | VIIa | I | ST1155 (CC101) | lukE-lukD, hla, hlg2 | sdrC, sdrD, sdrE, fib, clfB, ebpS, cna | ||||
| PVL | MMR-v | 55/F | Wound | XIIIa | II | ST1153 (CC1153) | lukE-lukD, hla, hlg2 | sec, sei, sel | sdrC, sdrD, sdrE, fib, clfB, ebpS | blaZ | ||
| − | MMR-g | 46/F | Wound | VIIa | I | ST2549 (CC45) | hla | sea, seg, sei, sem, sen, seo | sdrC, sdrD, sdrE, clfB, ebpS, cna | blaZ | AMP, GEN | |
| − | MMR-44B | 53/M | Wound | VIIc | I | ST59 (CC59) | hla, hlg2 | seb, sek, seq | sdrC, sdrD, sdrE, fib, clfB, ebpS | blaZ, ermB, aac(6′)-Ie-aph(2″)-Ia, ant(6)-Ia | AMP, GEN, ERY, CLI, LVX | |
| − | MMR-a | 43/M | Wound | VIIa | I | ST3075 (Singleton) | hla, hlg2 | sek | sdrC, sdrD, sdrE, fib, clfB, ebpS, cna | blaZ, ant(4′)-Ia, tet(K) | AMP, GEN, MIN | |
| − | MMR-14B | 20/M | Blood | Xa | II | ST2990 (Singleton) | lukE-lukD, hlg2 | sec, sel | sdrC, sdrD, sdrE, fib, ebpS, cna, edin-B | blaZ | AMP, ERY, CLI | |
| mecA, PVL | MMR-42A | 56/M | Wound | IV | IIIa | III | ST88 (CC88) | lukE-lukD, hlg2 | sei, sek, seq | sdrC, sdrD, sdrE, fib, clfB, ebpS | blaZ, tetK | OXA, AMP, MIN |
| mecA | MMR-55B | 25/F | High vaginal swab | IV | VIIb | I | ST59 (CC59) | hla, hlg2 | seb, sek, seq | sdrC, sdrD, sdrE, fib, clfB, ebpS | blaZ, ermB, aac(6′)-Ie-aph(2″)-Ia | OXA, AMP, GEN, ERY, CLI, LVX |
| mecA | MMR-57B | 66/F | Wound | IV | VIIb | I | ST59 (CC59) | hla, hlg2 | seb, sek, seq | sdrC, sdrD, sdrE, fib, ebpS | blaZ, ermB, aac(6′)-Ie-aph(2″)-Ia | OXA, FOX, AMP, GEN, ERY, CLI, LVX, SXT |
| mecA | MMR-22B | 20/F | Wound | IV | IVb | I | ST6 (CC6) | lukE-lukD, hla, hlg2 | sea | sdrC, sdrD, sdrE, fib, ebpS, cna | blaZ | OXA, AMP |
CC, clonal complex; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus; PVL, Panton-Valentine leukocidin; ST, sequence type.
The following genes were detected in all strains: clfA, eno, fnbA, fnbB, hld, hlg and hlg2. ebpS-v indicates ebpS gene with internal deletion as described previously [18].
The following genes were not detected in any strain: sed, see, seh, sej, sep, ser, ses, set, seu, eta, etb, etd and tst-1.
The following genes were undetectable in any strains: tet(L), tet(M), ermA, msrA, aph(3′)-IIIa, acc(6′)-Ii, acc(6′)-Im, ant(9)-Ia, ant(9)-Ib, ant(3″)-Ia, aph(2″)-Ib, aph(2″)-Ic and aph(2″)-Id.
See Table 3 footnotes for abbreviations of antimicrobial agents and breakpoints for resistance. None of the strains showed resistance to arbekacin, cefazolin, cefmetazole, flomoxef, fosfomycin, teicoplanin, linezolid and vancomycin.
Discussion
In the present study, prevalence and drug resistance of staphylococcal species and genetic traits of S. aureus were elucidated for clinical isolates in a tertiary hospital in Myanmar. Distinctive features in this study were the high prevalence and antimicrobial resistance trend of S. haemolyticus, the low rate of MRSA, and the high rate of PVL among MSSA.
Among CNS species, S. haemolyticus has been described as occasionally the second most frequent clinical isolates after S. epidermidis, causing primarily bloodstream infections associated with the use of central venous catheters [5]. In the present study, with a lower number of blood isolates (11%), the frequency of S. haemolyticus was comparable to S. aureus and higher than that of S. epidermidis, suggesting the significance of this species in skin infections as well. In agreement with the view of this species having a great capacity to develop resistance to multiple classes of antimicrobial agents [29], [30], a high mecA-positive rate associated with high resistance rates to various antimicrobial agents of S. haemolyticus was observed in the present study. Although the mecA-positive rate in S. aureus was still low, a high rate of methicillin-resistant S. haemolyticus as well as S. epidermidis may alert us to the potential increase of drug-resistant isolates among staphylococcal species, including MRSA.
In the present study, the detection rate of PVL genes among S. aureus was notably high (40%), which may be related to a high proportion of wound swabs and pus (67%) in the specimens examined. Detection of PVL genes in six different STs among 20 S. aureus isolates suggests dissemination of PVL phages to multiple clones, while only the dominant clone, ST121, appears to spread within hospitals. Strain MMR-42A is the first PVL-positive MRSA isolated in Myanmar, having SCCmec-IV and genetic types ST88/spa-t729/agr-III/coa-III. The ST88 MRSA with SCCmec IV or V has been reported in both community and hospital settings in Africa (mostly in East Africa; Tanzania and Madagascar) [31], [32], [33] and Asia (mostly in China) [15], [34], [35], and less frequently in Europe [36], [37], [38], exhibiting agr type III and various spa types, with t186 being dominant. The spa type t729 detected in strain MMR-42A is genetically closely related to t186 and was described also for ST88 MRSA isolates in Africa [38], suggesting close relatedness to the previously described ST88 clone. PVL is associated with a part of ST88 MRSA as well as MSSA. In Bangladesh and China, neighbouring countries to Myanmar, PVL-positive ST88 MSSA and/or MRSA was reported [34], [39], [40]. Detection of ST88 MRSA in Myanmar suggests the potential spread of this clone in Asia, and there should be concern in healthcare settings resulting from the presence of PVL in this clone.
ST121 MSSA, mostly belonging to agr-IV, are distributed worldwide (mainly Africa, Asia and Europe) as a common cause of SSTI, often associated with PVL, while MRSA with this ST is rare [41], [42]. In our previous study in Myanmar on S. aureus isolates from wound/pus, food poisoning and healthy adults [18], PVL genes were detected in only ST121 MSSA strains with coa-Va/agr-IV from wounds in hospitalized patients. In the present study, ST121 was dominant among PVL-positive isolates and showed genetically identical traits to those of previous MSSA strains in Myanmar. Characteristically, ST121 S. aureus in Myanmar has been previously revealed to harbour the genes of bone sialoprotein and a variant of elastin binding protein with 180 bp deletion [18], which was also found in PVL-positive ST121 MSSA in the present study. Therefore, we suggest that a single ST121 PVL-positive S. aureus clone has been persisting as a cause of SSTI in Myanmar. Although virulence of ST121 MSSA might be increased with PVL and other toxins, this clone is generally susceptible to most antimicrobial agents. Thus, active promotion of early detection and treatment is recommended for infections with this clone in healthcare settings.
It is of note that a novel staphylocoagulase genotype, coa XIII, was assigned to a PVL-positive MSSA strain (MMR-v) belonging to the rare ST1153, which was isolated from 55-year-old patient with wound infection. The D1 and D2 regions of staphylocoagulase which define the coa genotype (subtype) is considered to be responsible for antibody recognition as well as contact with prothrombin [43], [44]. Accordingly, genetic diversity of the D1/D2 regions is suggested to be caused by selection with antibody and/or prothrombin in the host. Hence, increased virulence is concerned with the emergence of S. aureus with the new coa type as a result of the absence of immune response against the novel antigen of the virulence factor. In the present study, ST1930 MSSA was resistant to erythromycin and levofloxacin, which is the distinctive feature of resistance among PVL-positive isolates. Although the significance of ST1930 MSSA is not evident, S. aureus with CC96, to which ST1930 belongs, is revealed to secrete variable to high levels of alpha toxin [45], suggesting relevance to the increased virulence. Therefore, the prevalence of the novel PVL-positive MSSA clones ST1153 and ST1930 should be carefully monitored in Myanmar.
Despite low MRSA rates among S. aureus in the present study, it was notable that ST59 was identified in two isolates with SCCmec-IV as well as an MSSA isolate. These isolates were PVL negative, however, resistant to multiple antimicrobial agents harbouring resistance genes such as erm(B). ST59 (CC59) MRSA has been classified into some groups [3], [42], with PVL-positive strains with SCCmec-V predominating in Taiwan and other Asian countries (Taiwan clone), SCCmec-IV-harbouring PVL-positive strains known as USA1000 clone mostly restricted to the United States and PVL-negative (or positive) MRSA with SCCmec-IV or V in Australia. The ST59 PVL-negative MRSA-IV, with the same genetic traits as the ST59 isolates in the present study, was also detected at a high rate in the nasal cavities of children in Taiwan [46]. Thus, we suggest that ST59 S. aureus may be distributed widely in Asia as well as Australia and may occasionally acquire SCCmec and/or PVL phage, associated with their clonal spread. In Myanmar, caution may be necessary for the ST59 MRSA in hospitals regarding acquisition of PVL genes and more drug resistance.
In summary, the present study elucidated drug resistance and genetic traits of clinical isolates of staphylococci in a tertiary-care hospital in Myanmar. Further studies are needed in this country to survey the prevalence of methicillin-resistant CNS, MRSA and PVL-positive S. aureus and their drug resistance for control of staphylococcal infections in healthcare settings.
Acknowledgement
This study was supported by a Grant-in-Aid for Scientific Research (KAKENHI) (26460804) from the Japan Society for Promotion of Science (JSPS).
Footnotes
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.nmni.2015.12.007.
Conflict of interest
None declared.
Appendix A. Supplementary data
The following is the supplementary data related to this article:
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