Utilization of Molecular Systems for the Identification and Typing of Clinically Relevant Multiple Drug Resistant Staphylococcus Aureus

Staphylococcus aureus is an important human nosocomial pathogen that can cause a variety of skin infections and toxin-mediated diseases including gastroenteritis, staphylococcal scalded-skin syndrome and toxic shock syndrome. Whilst the use of antibiotics can kill most strains of pathogenic microbes, the increase of multiple drug resistant S. aureus, especially among hospital patients have been a worrying trend. In order to treat patients infected with this organism at the earliest time possible and to prevent further nosocomial outbreaks, it is necessary to rapidly identify the multiple drug resistant S. aureus, so that the patient can be treated with the correct antibiotic on time and thus prevent further complications. S. aureus besides being a nosocomial pathogen causing frequent nosocomial infections also causes community acquired outbreaks. The increased prevalence of S. aureus infections may be prevented if the epidemiology of S. aureus is studied, whereby the spread of outbreak clones can be identified and treated with the appropriate drugs. Therefore, the aim of this study was to investigate suitable molecular systems for use in the rapid identification ofS. aureus and detection of multiple drug resistant S. aureus and for typing of strain variations for epidemiological understanding ofS. aureus in Malaysia. In this study, a total of eighty-nine clinical S. aureus isolates obtained from five different hospitals in Malaysia and from one pathology laboratory were studied. All the isolates were confirmed for S. aureus by the presence of species-specific Sa442 fragment (s. aureus specific fragment). Sequencing of the Sa442 fragment from isolates obtained from the different geographical locations identified this fragment as an epidemiological marker as it showed a wide variation of 1-10 % in the nucleotide sequences among the S. aureus isolates studied. For rapid identification of S. aureus and detection of multiple drug resistant strains, two molecular assays were utilized. In the first assay, a multiplex peR based strategy was used, whereby the genes responsible for methicillin (mecA), mupirocin (ites2), gentamycin (aac(6')-aph(2")), erythromycin(ermA) resistance and species specific Sa442 fragment were amplified. Results of the assay indicated the amplification of antibiotic resistant genes and Sa442 fragment at the expected sizes of 533,456, 174, 139 and 108bp respectively. All the amplified products were further confirmed by sequencing. The second assay was a membrane assay based on an optimized dot blot hybridization technique. In the dot blot assay, a set of oligonuclotide probes designed from the antibiotic resistance genes and the Sa442 fragment sequences were highly sensitive and specific for the respective bacterial target genes. The membrane assay developed was able to detect multiple drug resistant S. aureus isolates in less than two hours after obtaining pure culture isolates. Detection is also possible in less than two hours from spiked urine and blood samples, as well as for direct nasal samples. The results obtained with both polymerase chain reaction (PCR) and membrane assay were found to be similar whereby, 58.8, 67.7, 97.7 and 1.1% of the isolates carried mecA, aac(6')-aph(2"), ermA and iles-2 genes respectively. The overall correlation between the antibiotic resistance (disc diffusion test) and presence of antibiotic resistant genes (PCR and membrane assay) were found to be 77.3% for methicillin, 73.7 % for gentamycin, 95.4% for erythromycin and 100% mupirocin. The molecular epidemiology of local S. aureus was studied using randomly amplified polymerase chain reaction (RAPD) and repetitive element sequence based PCR (repPCR). Four out of the 20 arbitrary primers screened were highly efficient for use in molecular typing of S. aureus isolates. The rep-PCR typing primers designed from the staphylococcal repetitive sequences (STAR) were also markedly feasible for typing of S. aureus isolates. The RAPD study for molecular epidemiology showed wide variation in S. aureus isolates, as seen by genetic distance value based on Jaccard's index ranged from 0.037 to 0.954545. Similarly, in rep-PCR study, a wide variation in genetic distance value based on Jaccard's index ranged from 0.037037 to 0.894737. A wide variation in genetic distance was seen in the clonal diversity of local S. aureus isolates, where by, isolates were divided into four (4) clones namely Miri, Kuantan, Kota Bharu and Seremban, with Miri as the most predominant clone. In addition, RAPD was able to distinguish between methicillin resistant Staphylococcus aureus (MRSA) and non-MRSA isolates, showing the spread of two MRSA clones in Malaysia. RAPD analysis produced two (2) molecular markers, at positions 500 bp with primer OPAE 14 and 750 bp with OPAE 15, whereas, rep-PeR produced three (3) molecular markers, positions 500 with rep primer!, between 1500 and 2000 bp with rep 2, and slightly above 750 bp with rep primer 3, in most of the S. aureus isolates studied. From the five (5) markers obtained with RAPD and rep-PCR, the putative 500 bp rep marker was cloned in PCR 2.1 Topo vector and sequenced. The 500 bp rep marker was selected, as this marker was obtained through the amplification of S. aureus isolates with the primer designed from S. aureus genome and also because of the small size. The sequence obtained identified the repmarker as a 489 bp fragment, showing 95% homology to a region in glyceraldehydes -3phosphate dehydrogenase (GAP) operon in S. aureus genome, whose coding potential is unknown. The higher percentage (95%) similarity of the rep marker to S. aureus genome emphasized the importance of the rep marker in species-specific identification. To investigate the potentiality of the rep marker in species-specific identification of S. aureus isolates, a PCR and a membrane assay were developed with primers and probe designed from the rep sequence. The PCR and membrane assay showed positive signal for all eighty-nine S. aureus isolates tested and no signal was seen for other Grampositive and Gram-negative species tested, appreciating the specificity and the sensitivity of rep primers and probes in species-specific detection of S. aureus isolates. Sequencing of the rep marker from isolates obtained from different geographical locations, identified this marker (rep) as a potential diagnostic marker as it is highly conserved in S. aureu genome showing 98-99% sequence similarity among the isolates. Besides for diagnosis, using two typing procedures (RAPD and rep-peR) to study the clonal relatedness among the local S. aureus isolates which were developed with suitable RAPD and rep primers, they were able to correctly type S. aureus isolates according to the geographical location and also to differentiate between MRSA and non-MRSA. Although the RAPD primers are commercially available, the rep primers identified are still not published for use in typing S. aureus. The patterning of the novel rep primers and probe for typing and diagnosis of S. aureus will be extremely useful in the clinical diagnosis as the primers and probes innovated are highly specific and ubiquitous in all S. aureus isolates. These novel achievements made in the study will be of great value in the modern diagnostic era as the molecular systems optimized could be readily applied in the clinical diagnosis due to the specificity and rapidity in the detection of multiple drug resistant S. aureus. The achievements of the current study is especially a significant contribution to the clinical diagnostics and infectious disease research because the utilization of the optimized system incorporated as diagnostic kit will enhance the sensitivity and rapidity of molecular based detection in combination with sub-typing ability. Therefore, the routine application of the molecular systems optimized and the primers and probes developed in this study for the rapid identification of multiple drug resistant S. aureus and to study the epidemiology of S. aureus will definitely contribute towards early diagnosis of S. aureus infection in clinical laboratories. The epidemiological investigation will aid in developing more effective strategies in preventing and controlling the further spread of multiple drug resistant S. aureus clones in Malaysi

Long-term preservation of Leptospira spp.: challenges and prospects

Preservation of leptospiral cultures is tantamount to success in leptospiral diagnostics, research, and development of preventive strategies. Each Leptospira isolate has imperative value not only in disease diagnosis but also in epidemiology, virulence, pathogenesis, and drug development studies. As the number of circulating leptospires is continuously increasing and congruent with the importance to retain their original characteristics and properties, an efficient long-term preservation is critically needed to be well-established. However, the preservation of Leptospira is currently characterized by difficulties and conflicting results mainly due to the biological nature of this organism. Hence, this review seeks to describe the efforts in developing efficient preservation methods, to discover the challenges in preserving this organism and to identify the factors that can contribute to an effective long-term preservation of Leptospira. Through the enlightenment of the previous studies, a potentially effective method has been suggested. The article also attempts to evaluate novel strategies used in other industrial and biotechnological preservation efforts and consider their potential application to the conservation of Leptospira spp

Evaluation of diagnostic accuracy of loop-mediated isothermal amplification method (LAMP) compared with polymerase chain reaction (PCR) for Leptospira spp. in clinical samples: a systematic review and meta-analysis

Loop-mediated isothermal amplification (LAMP) test is widely used in molecular diagnostics as a point-of-care technique alternative to traditional PCR especially in resource-limited countries. LAMP has been recently used to diagnose leptospirosis. Therefore, we undertook a systematic review and meta-analysis to compare the accuracy of LAMP with PCR in the diagnosis of leptospirosis. Sixty-one studies were extracted from three international databases and analyzed throughout using the PRISMA guideline. The pooled sensitivity of LAMP and PCR technique was 0.80 (95% CI: 0.58–0.90) and 0.54 (95% CI: 0.35–0.67) respectively indicating that LAMP is more sensitive than PCR. The Q* value of LAMP and PCR-based technique is 274.61 and 397.95, respectively. Among the analyzed studies, significant heterogeneity was observed where I2 is 90.90% for LAMP-based and 86.18% for PCR-based. Our study suggests that LAMP has better diagnostic accuracy than PCR. However, future work should be carried out to reduce heterogeneity as well as to improve and develop effective intervention strategies

In vitro antibacterial and antibiofilm activities of chlorogenic acid against clinical isolates of stenotrophomonas maltophilia including the trimethoprim / sulfamethoxazole resistant strain

The in vitro antibacterial and antibiofilm activity of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia was investigated through disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill and biofilm assays. A total of 9 clinical S. maltophilia isolates including one isolate resistant to trimethoprim / sulfamethoxazole (TMP/SMX) were tested. The inhibition zone sizes for the isolates ranged from 17 to 29 mm, while the MIC and MBC values ranged from 8 to 16 g mL-1 and 16 to 32 g mL-1. Chlorogenic acid appeared to be strongly bactericidal at 4x MIC, with a 2-log reduction in viable bacteria at 10 h. In vitro antibiofilm testing showed a 4-fold reduction in biofilm viability at 4x MIC compared to 1x MIC values (0.085 < 0.397 A 490 nm) of chlorogenic acid. The data from this study support the notion that the chlorogenic acid has promising in vitro antibacterial and antibiofilm activities against S. maltophilia

Iron and virulence in Stenotrophomonas maltophilia: all we know so far

Stenotrophomonas maltophilia is a multi-drug-resistant global opportunistic nosocomial pathogen, which possesses a huge number of virulence factors and antibiotics resistance characteristics. Iron has a crucial contribution toward growth and development, cell growth and proliferation, and pathogenicity. The bacterium found to acquire iron for its cellular process through the expression of two iron acquisition systems. Two distinct pathways for iron acquisition are encoded by the S. maltophilia genome-a siderophore-and heme-mediated iron uptake system. The entAFDBEC operon directs the production of the enterobactin siderophore of catecholate in nature, while heme uptake relies on hgbBC and potentially hmuRSTUV operon. Fur and sigma factors are regulators of S. maltophilia under iron-limited condition. Iron potentially act as a signal which plays an important role in biofilm formation, extracellular polymeric substances (EPS), extracellular enzymes production, oxidative stress response, diffusible signal factor (DSF) and siderophore production in S. maltophilia. This review summarizes the current knowledge of iron acquisition in S. maltophilia and the critical role of iron in relation to its pathogenicity

The 'checkmate' for iron between human host and invading bacteria: chess game analogy

Iron is an essential nutrient for all living organisms with critical roles in many biological processes. The mammalian host maintains the iron requirements by dietary intake, while the invading pathogenic bacteria compete with the host to obtain those absorbed irons. In order to limit the iron uptake by the bacteria, the human host employs numerous iron binding proteins and withholding defense mechanisms that capture iron from the microbial invaders. To counteract, the bacteria cope with the iron limitation imposed by the host by expressing various iron acquisition systems, allowing them to achieve effective iron homeostasis. The armamentarium used by the human host and invading bacteria, leads to the dilemma of who wins the ultimate war for iron

Frequency of methicillin resistant Staphylococcus aureus in the noses of Malaysian chicken farmers and their chicken

The prevalence of methicillin resistant Staphylococcus aureus (MRSA) and methicillin sensitive Staphylococcus aureus (MSSA) carriage among poultry and poultry farmers in Malaysia is largely unknown. In the current investigation, chickens and chicken farmers from 30 chicken farms were screened for MRSA and S. aureus carriage. The genetic characteristics of the isolates were determined through multi locus sequence typing (MLST), Staphylococcus protein A (spa) typing and virulent gene profiling. The outcome of the study showed lack of MRSA and extremely low S. aureus prevalence (n=7 of 503, 1.4%) among chicken flocks and the poultry farmers in Malaysia. Staphylococcus aureus isolates belonged to 4 sequence types (ST): ST97 (spa type t359), ST1179 (t359), ST 692 (t2247) and ST188 (t189). It can be concluded that MRSA/MSSA prevalence is very low among chicken and chicken farmers, human and chicken cross transmission of S. aureus does not seem to be a threat in Malaysia

Genomic data of leptospira interrogans hp358 isolated from rodent captured from the human leptospirosis suspected areas of Selangor state, Malaysia

The data provided in this article is the genomic sequence of new Leptospira isolate, Leptospira interrogans strain HP358 ( L. interrogans HP358) isolated from rodent, Sundamys muel- leri (S. muelleri) , captured from the human leptospirosis sus- pected area, in forest environment, Hulu Perdik, Selangor. The kidney of the rodent was cultured, and the genomic DNA of pure Leptospira isolate was extracted and sequenced. The de novo assembly of genome generated 118 contigs with N50 of 133,176bp. The genome size of the L. interrogans HP358 was determined with a length of 4,808,724 and 35.01% G + C content with 229 subsystems, 5236 coding sequences and 39 RNAs. The whole genome shotgun project has been de- posited in NCBI GenBank under the accession number JAF- CYY0 0 0 0 0 0 0 0 0.1

Antimicrobial susceptibility profiles, serotype distribution and virulence determinants among invasive, non-invasive and colonizing Streptococcus agalactiae (group B streptococcus) from Malaysian patients

A total of 103 group B streptococci (GBS) including 22 invasive, 21 non-invasive, and 60 colonizing isolates were collected in a Malaysian hospital (June 2010–October 2011). Isolates were characterized by conventional and molecular serotyping and analyzed for scpB, lmb, hylB, cylE, bac, bca and rib gene content. Antimicrobial susceptibility to penicillins, macrolides, lincosamides, quinolones and tetracyclines was determined using disk diffusion and the MICs for penicillin were determined by E-test. Molecular serotyping for all eight serotypes (Ia, Ib, II–VII) was in full accordance with conventional serotyping. Overall, taking CS and MS together, serotype VI was the most common capsular type (22.3 %) followed by VII (21.4 %), III (20.4 %), Ia (17.5 %), V (9.7 %), II (7.7 %) and IV (1 %). Susceptibility to beta-lactam antimicrobials was prevalent (100 %). Resistance rates for erythromycin, clindamycin and tetracycline were 23.3 %, 17.5 % and 71.8 %, respectively. PCR-virulence gene screening showed the presence of cylE, lmb, scpB and hylB in almost all the isolates while rib, bca, and bac genes were found in 29.1 %, 14.6 % and 9.7 % of the isolates. Certain genes were significantly associated with specific serotypes, namely, rib with serotypes Ia, II, III and VI; bca and bac with serotypes II and III. Furthermore, serotype Ia was significantly more common among patients with invasive infections (p < 0.01) and serotype VI isolates were significantly more common among carriers (p < 0.05). In summary, serotype distribution correlates with virulence gene content will be useful in epidemiological studies and design of vaccines