We investigated the potential of USA300 MRSA emergence in Malaysia by examining 268 MSSA isolates from both community (110) and healthcare (158) settings. Nine isolates from both the environments were similar to the USA300 MRSA background based on MLST, spa and PFGE type. These results underscore the importance of continued surveillance to monitor the emergence of USA300 MRSA in Malaysia.
Methicillin-resistant Staphylococcus aureus (MRSA) from Malaysia were shown to possess staphylococcal cassette chromosome mec (SCCmec)-III and IIIA. Spa sequencing and multi-locus sequence typing (MLST) documented t037 and ST 239 (CC8) for 83.3% of the isolates. This confirms observations in several other Far Eastern countries and corroborates the epidemicity of this clone.
The humoral immune response against 43 staphylococcal antigens was compared among hospitalized patients where none of them had any staphylococcal infection on the day of admission with or without nasal Staphylococcus aureus carriage. Fifty-nine carriers and 59 matched non-carriers were studied. The carriers harbored S. aureus of 35 different spa types, including three t037/ST239 methicillin-resistant S. aureus (MRSA) (5.1%). Among the 118 patients, 31 acquired S. aureus during hospitalization. In colonized and non-colonized patients, unique patterns of S. aureus-specific immune responses were observed. The mean fluorescence indices (MFIs) of antibodies against 36/43 (83.7%) antigens were seen to be elevated among carriers. The MFI among carriers with acquisition was significantly higher for staphylococcal superantigen-like protein 5 (SSL5, p = 0.028) when compared to carriers without acquisition. High antibody levels against staphylococcal enterotoxin A (SEA) among carriers illustrate its role as a superantigen in both infection and colonization. We also report a dynamic immune response in S. aureus-carrying patients against the recently reported formyl peptide receptor-like inhibitory (FLIPr)-like protein. In the current study, the dynamics of antibodies against staphylococcal antigens among carrier patients seem quite similar to non-carrier patients. To better understand the dynamic immunogenicity during S. aureus infection and colonization, artificial colonization studies and investigation of the changes in the levels of antibodies against other staphylococcal antigens are recommended.
The prevalence and spread of mupirocin and antiseptic resistance among colonizing and infectious Staphylococcus aureus were determined. S. aureus isolated from anterior nares and infection sites of patients hospitalized in the largest tertiary care referral hospital in Malaysia was investigated for mupirocin and antiseptic susceptibility testing, and for PCR detection of mupA, qacA/B, and smr genes. Twelve isolates showed resistance to mupirocin by disk diffusion, of which 10 (3.8%) harbored the mupA gene. Minimum inhibitory concentrations (MICs) ranged from 64 to 768 μg/ml for mupA positive and below 46 μg/ml for negative isolates. The mupA was more common among ST239 isolates (70%). The qacA/B was carried in 67 out of 95 methicillin-resistant Staphylococcus aureus (MRSA) (70.5%) and 3 out of 164 methicillin-susceptible Staphylococcus aureus (MSSA) (1.8%), while smr was carried in 6 out of 95 MRSA (6.3%) strains. MICs ranged from 3.9 to 15.6 μg/ml for benzethonium chloride (BTC) and benzalkonium chloride (BKC), and from 10.3 to 20.7 μg/ml for chlorhexidine digluconate (CHG). Isolates with qacA/B and smr or qacA/B alone showed higher MIC (20.7 μg/ml for CHG and 15.6 μg/ml for BTC and BKC) than the isolates that lacked antiseptic resistance genes (10.3 μg/ml for CHG and 3.9 μg/ml for BTC and BKC). In 16 cases, ST239 was isolated from the infection site and the nares simultaneously, and shared identical resistance patterns (qacAB or qacAB+smr), suggesting possible endogenous infection. Spread of low-level mupirocin resistance expressing ST239 MRSA and high-level resistance expressing emerging ST1, co-existing with antiseptic-resistant genes showing elevated MICs, should be monitored for effective infection control.
Methicillin-resistant Staphylococcus aureus (MRSA) is well known for its epidemicity, with the emergence of new clones on a daily basis. Diversity in the clonal types of MRSA challenges the success of treatment, as different clones respond to different sets of antibiotics. However, the antibiotic susceptibility among the isolates within the same clones is largely unexplored. In a previous study on MRSA epidemiology in Malaysia, we identified six major clonal complexes (ST-239-CC8, ST-1-CC1, ST-188-CC1, ST-22-CC22, ST-7-CC7 and ST-1283-CC8). In the present study, we investigated the antibiotic susceptibility patterns of isolates of different clones. Three hundred and eighty-nine MRSA isolates were subjected to the disc diffusion test, oxacillin minimum inhibitory concentration (MIC) determination and assessment of the distribution of macrolide, lincosamide and streptogramin B (MLS(B)) resistance genes. Thirty-six different antibiotic profiles were observed: 30 (83.3 %) among ST-239, 2 (5.6 %) among ST-1283 and 1 (2.8 %) each for ST-1, ST-7, ST-22 and ST-188. All ST-239 (362, 9 %) isolates were multiple drug-resistant (MDR; resistant to more than three classes of antibiotics) and had oxacillin MICs >256 mg/l. Among the 385 clindamycin-resistant isolates, 375 (96.4 %) illustrated inducible resistance (D-zone-positive), while 10 (2.6 %) showed constitutive resistance. The vast majority of the macrolide-resistant isolates carried the ermA gene (95.1 %), followed by ermC (12.9 %). Diversity in the antibiotic susceptibilities of isolates within the clones emphasises the need for continuous surveillance of MDR strains to prescribe the correct antibiotic rather than empirical treatment. This will likely reduce the emergence of new endemic or epidemic resistant MRSA clones.
The humoral immune responses against 46 different staphylococcal antigens in 27 bacteremia patients infected by clonally related methicillin-resistant Staphylococcus aureus (MRSA) strains of a single sequence type (ST) 239 were investigated. A group of non-infected patients (n = 31) hospitalized for different reasons served as controls. All strains were confirmed as ST 239 by S. aureus and mecA-specific PCR, spa, and multi-locus sequence typing (MLST). In each bacteremia patient, a unique pattern of S. aureus antigen-specific immune responses after infection was observed. Antibody levels among bacteremia patients were significantly higher than controls for HlgB (P = 0.001), LukD (P = 0.009), LukF (P = 0.0001), SEA (P = 0.0001), SEB (P = 0.011), SEC (P = 0.010), SEQ (P = 0.049), IsaA (P = 0.043), IsdA (P = 0.038), IsdH (P = 0.01), SdrD (P = 0.001), SdrE (P = 0.046), EsxA (P = 0.0001), and SA0104 (P = 0.0001). On the other hand, the antibody levels were significantly higher among controls for SSL3 (P = 0.009), SSL9 (P = 0.002), and SSL10 (P = 0.007) when the IgG level on the day of infection was compared with that measured on the day of admission. Diversity was observed in the immune response against the antigens. However, a set of antigens (IsaA, IsdA, IsdH, SdrD, and HlgB) triggered a similar type of immune response in different individuals. We suggest that these antigens could be considered when developing a multi-component (passive) vaccine. SEA and/or its specific antibodies seem to play a critical role during ST239 MRSA bacteremia and SEA-targeted therapy may be a strategy to be considered.
We performed a prospective observational study in a clinical setting to test the hypothesis that prior colonization by a Staphylococcus aureus strain would protect, by colonization interference or other processes, against de novo colonization and, hence, possible endo-infections by newly acquired S. aureus strains. Three hundred and six patients hospitalized for >7 days were enrolled. For every patient, four nasal swabs (days 1, 3, 5, and 7) were taken, and patients were identified as carriers when a positive nasal culture for S. aureus was obtained on day 1 of hospitalization. For all patients who acquired methicillin-resistant S. aureus (MRSA) or methicillin-susceptible S. aureus via colonization and/or infection during hospitalization, strains were collected. We note that our study may suffer from false-negative cultures, local problems with infection control and hospital hygiene, or staphylococcal carriage at alternative anatomical sites. Among all patients, 22% were prior carriers of S. aureus, including 1.9% whom carried MRSA upon admission. The overall nasal staphylococcal carriage rate among dermatology patients was significantly higher than that among neurosurgery patients (n = 25 (55.5%) vs. n = 42 (16.1%), p 0.005). This conclusion held when the carriage definition included individuals who were nasal culture positive on day 1 and day 3 of hospitalization (p 0.0001). All MRSA carriers were dermatology patients. There was significantly less S. aureus acquisition among non-carriers than among carriers during hospitalization (p 0.005). The mean number of days spent in the hospital before experiencing MRSA acquisition in nasal carriers was 5.1, which was significantly lower than the score among non-carriers (22 days, p 0.012). In conclusion, we found that nasal carriage of S. aureus predisposes to rather than protects against staphylococcal acquisition in the nose, thereby refuting our null hypothesis.
Leptospirosis is an important zoonotic bacterial disease caused by Leptospira spp. Earlier studies from North Khorasan province (Iran) reported the presence of Leptospira in wild canines and rodents. To date, there is no data on the seroprevalence of leptospirosis among humans in this province. This study was performed to determine the prevalence of human leptospiral infection among people with different occupations. The study was conducted in urban and rural areas of the province. Among the serum samples collected from 278 subjects, 3 (1.1%) showed positive reaction with titer of 1:100 by the microscopic agglutination test (MAT). Positive reactions were detected against Leptospira interrogans Canicola and L. interrogans icterohemorrhagic and all these samples were from livestock farmers (n = 3/106, 2.7%). The current study revealed that, though Leptospira infection is low in North Khorasan province, regular monitoring of the livestock and the farmers are important.
Despite the association of methicillin-susceptible S. aureus (MSSA) with several life-threatening diseases, relatively little is known about their clinical epidemiology in Malaysia. We characterized MSSA isolates (n=252) obtained from clinical and community (carriage) sources based on spa sequencing and multilocus sequence typing (MLST). The prevalence of several important virulence genes was determined to further define the molecular characteristics of MSSA clones circulating in Malaysia. Among the 142 clinical and 110 community-acquired MSSA isolates, 98 different spa types were identified, corresponding to 8 different spa clonal clusters (spa-CCs). In addition, MLST analysis revealed 22 sequence types (STs) with 5 singletons corresponding to 12 MLST-CCs. Interestingly, spa-CC084/085 (MLST-CC15) (p=0.038), spa-non-founder 2 (MLST-ST188) (p=0.002), and spa-CC127 (MLST-CC1) (p=0.049) were identified significantly more often among clinical isolates. spa-CC3204 (MLST-CC121) (p=0.02) and spa-CC015 (MLST-CC45) (p=0.0002) were more common among community isolates. Five dominant MLST-CCs (CC8, CC121, CC1, CC45, and CC5) having clear counterparts among the major MRSA clones were also identified in this study. While the MSSA strains are usually genetically heterogeneous, a relatively high frequency (19/7.5%) of ST188 (t189) strains was found, with 57.8% of these strains carrying the Panton-Valentine leukocidin (PVL). Analysis of additional virulence genes showed a frequency of 36.5% and 36.9% for seg and sei and 0.8% and 6.3% for etb and tst genes, respectively. Arginine catabolic mobile element (ACME) was detected in 4 community isolates only. These represent the first isolates harbouring this gene in an Asian region. In conclusion, MSSA from the Malaysian community and their clinical counterparts are genetically diverse, but certain clones occur more often among clinical isolates than among carriage isolates and vice versa.
We define the epidemiology of predominant and sporadic methicillin-resistant Staphylococcus aureus (MRSA) strains in a central teaching and referral hospital in Kuala Lumpur, Malaysia. This is done on the basis of spa sequencing, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and virulence gene profiling. During the period of study, the MRSA prevalence was 44.1%, and 389 MRSA strains were included. The prevalence of MRSA was found to be significantly higher in the patients of Indian ethnicity (P < 0.001). The majority (92.5%) of the isolates belonged to ST-239, spa type t037, and possessed the type III or IIIA SCCmec. The arginine catabolic mobile element (ACME) arcA gene was detected in three (1.05%) ST-239 isolates. We report the first identification of ACME arcA gene-positive ST-239. Apart from this predominant clone, six (1.5%) isolates of ST-22, with two related spa types (t032 and t4184) and a singleton (t3213), carrying type IVh SCCmec, were detected for the first time in Asia. A limited number of community-acquired (CA) MRSA strains were also detected. These included ST-188/t189 (2.1%), ST-1/t127 (2.3%), and ST-7/t091 (1%). Panton-Valentin leukocidin (PVL) was detected in all ST-1 and ST-188 strains and in 0.7% of the ST-239 isolates. The majority of the isolates carried agr I, except that ST-1 strains were agr III positive. Virulence genes seg and sei were seen only among ST-22 isolates. In conclusion, current results revealed the predominance of ST-239-SCCmec III/IIIA and the penetration of ST-22 with different virulence gene profiles. The emergence in Malaysia of novel clones of known epidemic and pathogenic potential should be taken seriously.
Clinical information about genotypically different clones of biofilm-producing Staphylococcus aureus is largely unknown. We examined whether different clones of methicillin-sensitive and methicillin-resistant S. aureus (MSSA and MRSA) differ with respect to staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in biofilm formation. The study used 60 different types of spa and determined the phenotypes, the prevalence of the 13 MSCRAMM, and biofilm genes for each clone. The current investigation was carried out using a modified Congo red agar (MCRA), a microtiter plate assay (MPA), polymerase chain reaction (PCR), and reverse transcriptase polymerase chain reaction (RT-PCR). Clones belonging to the same spa type were found to have similar properties in adheringto the polystyrene microtiter plate surface. However, their ability to produce slime on MCRA medium was different. PCR experiments showed that 60 clones of MSSA and MRSA were positive for 5 genes (out of 9 MSCRAMM genes). icaADBC genes were found to be present in all the 60 clones tested indicating a high prevalence, and these genes were equally distributed among the clones associated with MSSA and those with MRSA. The prevalence of other MSCRAMM genes among MSSA and MRSA clones was found to be variable. MRSA and MSSA gene expression (MSCRAMM and icaADBC) was confirmed by RT-PCR.