Suwanna Trakulsomboon

Staphylococcal Cassette Chromosome mec (SCCmec) Typing of Methicillin-Resistant Staphylococcus aureus Strains Isolated in 11 Asian Countries: a Proposal for a New Nomenclature for SCCmec Elements

ABSTRACT A description of staphylococcal cassette chromosome mec (SCCmec) elements carried by 615 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 11 Asian countries is reported, and a novel nomenclatural system based on their structures is proposed. The 615 strains were classified as type 3A (370 strains), type 2A (207 strains), type 2B (32 strains), type 1B (1 strain), and nontypeable (5 strains). The previously reported type III SCCmec (DDBJ/EMBL/GenBank accession no. AB037671) carried by the MRSA strain 85/2082 was ascertained to be composed of two SCC elements, type 3A SCCmec and SCCmercury. PCR analysis indicated that 310 of 370 type 3A SCCmec strains carried both SCC elements. These strains were prevalent in eight countries: Thailand, Sri Lanka, Indonesia, Vietnam, Philippines, Saudi Arabia, India, and Singapore. The remaining 60 type 3A SCCmec strains differed with respect to the left extremity polymorphism or to the presence of ccrC. Among these, two were identified as carrying only type 3A SCCmec elements, but their left extremities differed. Type 2A SCCmec strains predominated in Korea and Japan, although the frequency of the presence of ant(4′)-1 gene downstream of mecA varied (53% for Korean strains; 93% for Japanese strains). Various SCCmec elements were identified in the tested strains, and limited numbers were identified by their multilocus sequence typing genotypes. These data suggest that numerous MRSA clones are disseminated in Asian hospitals, and these consist of minor clones that are presumed to have arisen locally and major clones that are presumed to have been introduced from other countries.

First report of methicillin-resistant Staphylococcus aureus with reduced susceptibility to vancomycin in Thailand.

ABSTRACT To investigate whether there are methicillin-resistantStaphylococcus aureus (MRSA) strains with reduced susceptibility to vancomycin in Thailand, a total of 155 MRSA strains isolated from patients hospitalized between 1988 and 1999 in university hospitals in Thailand were tested for glycopeptide susceptibility. All the strains were classified as susceptible to vancomycin and teicoplanin when judged by NCCLS criteria for glycopeptide susceptibility using the agar dilution MIC determination. Vancomycin MICs at which 50 and 90% of the isolates tested were inhibited (MIC50 and MIC90, respectively) were 0.5 and 1 μg/ml, respectively, with a range of 0.25 to 2 μg/ml. For teicoplanin, MIC50 and MIC90 were 2 μg/ml, with a range of 0.5 to 4 μg/ml. However, one-point population analysis identified three MRSA strains, MR135, MR187, and MR209, which contained subpopulations of cells that could grow in 4 μg of vancomycin per ml. The proportions of the subpopulations were 2 × 10−4, 1.5 × 10−6, and 4 × 10−7, respectively. The subsequent performance of a complete population analysis and testing for the emergence of mutants with reduced susceptibility to vancomycin (MIC ≥ 8 μg/ml) confirmed that these strains were heterogeneously resistant to vancomycin. Two of these strains caused infection that was refractory to vancomycin therapy. Pulsed-field gel electrophoresis showed that the two strains had identical SmaI macrorestriction patterns and that they were one of the common types of MRSA isolated in the hospital. This is the first report of heterogeneous resistance to vancomycin in Thailand and an early warning for the possible emergence of vancomycin resistance in S. aureus in Southeast Asia.

Ribotype differences between clinical and environmental isolates of Burkholderia pseudomallei

Burkholderia pseudomallei is isolated frequently from the soil in regions where the disease melioidosis occurs. However, recent surveys in Thailand have shown that the frequency of isolation of the organism from soil samples is not directly related to the incidence of melioidosis in an area. To determine whether strain populations of B. pseudomallei prevalent in soil are gentypically related to strains causing clinical disease, rRNA BamHI restriction fragment length polymorphisms (RFLP) of 139 soil environmental isolates and 228 human isolates were compared. Two groups of ribotype patterns were found. Group I comprised 37 different ribotype patterns which were characterised by five to eight hybridisation bands of 2.8- > 23 kb. All of these ribotypes were identified among the clinical isolates, and 18 of them were also found in 59 environmental isolates. Group II was represented by 12 ribotypes found only in environmental strains. These ribotype patterns comprised one to five bands in the size range 9- > 23 kb. All but one of the 73 isolates in this group grew on a minimal medium supplemented with L-arabinose. In contrast, only 3% of the 66 isolates from the environment with group I ribotype patterns could utilise this sugar as their sole energy source. These findings suggest that B. pseudomallei strains that utilise arabinose constitute a population that is genetically distinct from other environmental and clinical strains.

Molecular phylogeny of Burkholderia pseudomallei.

In terms of population structure, the species Burkholderia pseudomallei contains both clonal and non-clonal elements. By indexing variation in rRNA loci using the restriction endonuclease BamHI, we found that two ribotypes (types 1 and 3) are predominant in nature. Ribotype 3 is prevalent in Asian countries while ribotype 1 is more widespread. Some disease association was suggested for 4 ribotypes and strains of ribotype 4 were markedly associated with a fatal outcome. DNA macrorestriction (XbaI) profiles resolved by pulsed-field gel electrophoresis revealed great heterogeneity within the prevalent ribotypes and these profiles appeared to be reliable strain markers. Arabinose environmental strains were characterised by BamHI ribotypes that were markedly distinct form clinical and environmental isolates of the arabinose negative phenotype.

In Vitro Activity of Tigecycline against Burkholderia pseudomallei and Burkholderia thailandensis

ABSTRACT Investigation of the in vitro activity of tigecycline against Burkholderia pseudomallei and Burkholderia thailandensis revealed that the inhibition zone diameters of tigecycline against all isolates were ≥20 mm and that the MIC50 values were 0.5 and 1 μg/ml and the MIC90 values were 2 and 1.5 μg/ml for B. pseudomallei and B. thailandensis, respectively.

Recurrent Melioidosis: Possible Role of Infection with Multiple Strains of Burkholderia pseudomallei

We were interested to read the paper by Maharjan et al. suggesting that reinfection is more common than previously realized in melioidosis (1). There is one other possibility that they did not consider, which might account for isolates from recurrent melioidosis being genotypically distinct from isolates in primary infection, namely, initial infection with more than one genotype. We report here studies that suggest that this is a not infrequent occurrence. n nEighteen patients with culture-positive melioidosis seen in Ubon Ratchathani, northeastern Thailand, were studied. Fourteen patients yielded the organism from blood, three presented with localized abscesses with no systemic spread, and one had a urinary tract infection. Sets of 10 to 40 colonies (dependent on the total number of colonies per plate) were selected from primary culture plates of specimens from multiple body sites or from subculture of blood culture broths. Isolates were assigned to a BamHI ribotype pattern, and pulsed-field gel electrophoresis (PFGE) of XbaI DNA digests was used to discriminate further within a ribotype (4). Strains were then allocated a ribotype number or PFGE letter as part of a larger study (3). This showed that 13 of 18 patients were infected with a single strain. In contrast, five patients harbored more than one genotype (Table u200b(Table1).1). Four patients had two distinct genotypes detected, while one (A/C 383) had three. Different genotypes were detected both within a single specimen and from the same patient in samples collected on different days. Figure u200bFigure11 illustrates the DNA profiles of the isolates from patient U1128. All of the 10 colonies from blood cultures were homogeneous, and the same strain was one of two recovered from a swab of a toe wound in this patient. n n n nFIG. 1. n n(A) BamHI ribotype patterns of B. pseudomallei isolates. Lanes 1 and 4, toe wound; lanes 2, 3, and 5, blood. (B) XbaI restriction patterns obtained by PFGE. n n n n n nTABLE 1. n nVariation in genotypes of B. pseudomallei from 5 of 18 patients with melioidosis n n n nA statistical comparison (chi-square test) of the clinical manifestations and outcomes of patients infected with single versus multiple strains revealed that the latter were more associated with an acute presentation (5/13 versus 4/4), septicemia (5/12 versus 4/5), and a fatal outcome (6/13 versus 4/5) (P < 0.001), suggesting the possibility that this reflected an acute exposure to a large inoculum. n nInfection with B. pseudomallei is thought to result from exposure to organisms in the environment. Relatively little is known about the occurrence of different genotypes of B. pseudomallei in the environment, but it is not unreasonable to propose that several different strains may coexist within the complex ecosystem represented by paddy field mud, for example. If so, then concurrent infection with more than one strain might thus be expected to occur on occasion. Infection with multiple strains of the same species is not unknown and was previously described in melioidosis by Sexton et al. (2), who reported the presence of two different ribotypes at different body sites of a single patient. The fact that the proportion of cases of recurrent melioidosis caused by strains with a genotype different from that of the primary isolate increases as the time between episodes increases, as reported by Maharjan et al. (1), does indeed support their suggestion that such cases represent reinfection rather than relapse. However, the finding here that 27% of patients harbored different strains concurrently accords with the frequency of reinfection (25%) reported by Maharjan et al. (1) and might provide an alternative explanation for the appearance of apparently new strains over time.