Antibiotic resistance and molecular typing of clinical Staphylococcus aureus isolates from Malaysian military hospital

Objective:To determine the antibiotic resistance profile(ARP)of Staphylococcus(S.)aureus isolates and molecular typing of the methicillin-resistant S.aureus(MRSA)isolates from Tuanku Mizan Armed Forces Hospital(TMAFH),Kuala Lumpur.Methods:The ARP and presence of the pvl gene were determined for 209 S.aureus isolates from clinical specimens.Of these,123 were methicillin-susceptible S.aureus(MSSA)isolates and 86 were MRSA isolates.All MRSA isolates were characterized using SCCmec typing and spa typing.Descriptive analysis was performed to compare the demographic data with the phenotypic and genotypic variables of the S.aureus isolates.Results:No vancomycin-intermediate and-resistant S.aureus(VISA and VRSA,respectively)were detected among the study isolates.The MSSA isolates showed low resistance rates to all tested antibiotics,were commonly invasive(28/42,66.7%),and mostly harboured pvl(35/42,83.3%).Meanwhile,MRSA isolates showed high resistance to penicillin(86/86,100%),ampicillin(86/86,100%),sulbactam/ampicillin(86/86,100%),cefuroxime(81/86,94.19%),cefoperazone(76/86,88.37%),azithromycin(56/86,65.12%),and erythromycin(54/86,62.79%).The majority of MRSA isolates were of SCCmec type IVh(65/86,75.58%),spa type t032(55/85,63.95%),and grouped into spaCC-t022(66/85,77.65%).The t032 type was found to be associated with resistance traits to azithromycin and erythromycin(P<0.05).We also found several spa types that are typically associated with hospital-,community-,and livestock-associated MRSA co-existing in our MRSA population.Conclusions:This study reflected the consistent absence of VISA and VRSA and corroborated the clonal shifting of MRSA isolates in the Malaysian MRSA isolates.

Antimicrobial resistance and molecular typing of Salmonella in the chicken production chain in Hubei Province, China

Salmonella is a significant foodborne zoonotic pathogen that endangers both human and animal health.The goal of this research is to gain a preliminary understanding of Salmonella contamination and antimicrobial resistance in the chicken production chain in Hubei Province,China.1149 animal and environmental samples were collected from chicken farms,slaughterhouses,and retail markets in six cities across Hubei Province in China from 2019 to 2020,yielding Salmonella isolation rates of 4.68%(28/598),12.21%(47/385),and 9.64%(16/166),respectively.Seventeen distinct serotypes were detected among 53 non-clonal Salmonella strains,of which Meleagridis(26.42%,14/53)was the dominant serotype.Almost half of the strains(49.06%,26/53)were multi-drug resistant(MDR).Whole-genome sequencing(WGS)showed that 10 resistance genes(tetA,bla_(TEM),parC,qnrs1,floR,aac(6'-ly,aph(6)-ld,aph(3")-b,aac(6')-laa and sul2)and 7 categories of virulence genes were present in all three links in 22 non-clonal dominant serotype strains.It was shown that Salmonella in the chicken production chain in Hubei Province had a high resist-ance rate to Tetracycline(TET,73.58%),Ofloxacin(OFL,69.81%),Florfenicol(FFC,60.38%)and Ampicillin(AMP,39.62%)which was consistent with the widespread use of these drugs in the husbandry industry in China.Salmonella ST types determined by MLST and serotypes determined by WGS had a one-to-one correlation.Minimum spanning tree analysis revealed that there was cross contamination of Salmonella in farms and slaughterhouses,slaughterhouses and markets,animal samples and environmental samples.This work provides useful information for the prevention and control of contamination and antimicrobial resistance of Salmonella in the chicken production chain,as well as demonstrating the dependable role of WGS in Salmonella molecular typing.

Mycobacteria Interspersed Repetitive Units-Variable Number of Tandem Repeat, Spoligotyping and Drug Resistance of Isolates from Pulmonary Tuberculosois Patients in Kenya

Background: Molecular typing allows a rapid and precise species differentiation and is essential in investigating the spread of specific genotypes and any relationship with drug resistance. Methodology: To compare the discrimination power of 24-loci Mycobacteria interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) to spoligotyping in determining the circulating genotypes of Mycobacterium tuberculosis in isolates from pulmonary tuberculosis patients in Kenya, a total of 204 isolates were typed. Results: Spoligotyping identified 22 spoligo lineages;while 36(17.6%) isolates were not determined. MIRU-VNTR typing identified 12 genotypes;Kenya H37_Rv_ like, S-like that had never been reported before and which were not identified by spoligotyping were identified. Others were Uganda I and II, LAM, Beijing, TUR, EAI, Delhi/C, S and Haarlem. Only 8 (3.9%) were not defined by MIRU-VNTR. Delhi/CAS, EAI, S, S-like, LAM and Beijing had strains that showed resistance to all the five drugs tested. Two strains of EAI and 2 of S genotypes were resistant to all the five drugs tested. Beijing genotype commonly associated with drug resistance was found to be third in drug resistance (14.7%) after Delhi/CAS (28.9%) and LAM (17.6%) with the highest resistance towards isoniazid and pyrazinamide (3.9% each). MIRU-VNTR typing was more discriminative than spoligotyping;identifying 10 unique H37_Rv-like isolates designated KeniaH37_Rv_like genotype and 14 S-like genotype. Conclusion: MIRU-VNTR typing has not been reported in any other study in Kenya and its higher discrimination can help identify genotypes that cannot be determined by spoligotyping. Association of Beijing genotype drug resistance particularly isoniazid should be of concern since it may result in multidrug resistance in the patients.

Identification and Typing of Respiratory Adenoviruses in Guangzhou, Southern China Using a Rapid and Simple Method

Human adenoviruses (HAdVs), especially HAdV-B3, -E4 and -B7, are associated with Acute Respiratory Disease in Chinese children, and occasionally in adults. In order to establish and document the profiles of the respiratory adenovirus pathogen among children in Guangzhou, Southern China, a rapid, simple and practical method for identification and typing of respiratory adenoviruses was developed and evaluated. One pair of universal PCR primers was designed according to the conserved region of the hexon gene, which can detect not only HAdV-B3, -E4 and -B7, but also HAdV-B14, -F40 and -F41, with a specific 300bp PCR product. Three pairs of type-specific PCR primers were also designed according to the hypervariable regions of the hexon gene to type HAdV-B3, -E4 and -B7 by three independent PCR reactions, making it easy to optimize the PCR conditions. By using this method, one hundred throat swab specimens collected during Oct 2010 to Dec 2011 and suspected of being positive for adenoviral infection were identified and typed for adenoviruses. Of these samples, fifty-five were adenovirus-positive. The most common HAdV type was HAdV-B3, identified in 92.7% of samples, which is not only consistent with the data reported in 2004-2006, but also consistent with the recent report in Hangzhou, eastern China, indicating that HAdV-B3 has been circulating in Guangzhou, and maybe in eastern China, for many years. The method for the respiratory adenovirus identification and typing we developed is rapid, simple and practical, which has a potential in the real-time surveillance of circulating adenovirus strains and also to provide etiological evidence for the adenovirus-relative disease control and prevention in China.

Molecular Typing of Brucella Suis Collected from 1960s to 2010s in China by MLVA and PFGE

Brucellosis is a bacterial anthropozoonosis usually caused by Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis. Brucella suis, the causative agent of swine brucellosis, is classified into five biovars and preferentially infects different animal hostsIll. In China, brucellosis is a national notifiable communicable disease both in animals and in human. In 2009, 35 816 brucellosis cases were reported. The annual incidence was 2.7 per 100 000 population.

Human leukocyte antigen typing and crossmatch:A comprehensive review

Renal transplantation remains the best option for patients suffering from end stage renal disease(ESRD).Given the worldwide shortage of organs and growing population of patients with ESRD,those waitlisted for a transplant is ever expanding.Contemporary crossmatch methods and human leukocyte antigen(HLA) typing play a pivotal role in improving organ allocation and afford better matches to recipients.Understanding crossmatch as well as HLA typing for renal transplantation and applying it in clinical practice is the key step to achieve a successful outcome.Interpretation of crossmatch results can be quite challenging where clinicians have not had formal training in applied transplant immunology.This review aims to provide a worked example using a clinical vignette.Furthermore,each technique is discussed in detail with its pros and cons.The index case is that of a young male with ESRD secondary to Lupus nephritis.He is offered a deceased donor kidney with a 1-0-0 mismatch.His complement dependent cytotoxicity(CDC) crossmatch reported positive for B lymphocyte,but flow cytometry crossmatch(FCXM) was reported negative for both B and T lymphocytes.Luminex-SAB(single antigen bead) did not identify any donor specific antibodies(DSA).He never had a blood transfusion.The positive CDCcrossmatch result is not concordant with DSA status.These implausible results are due to underlying lupus erythematosus,leading to false-positive B-lymphocyte crossmatch as a result of binding immune complexes to Fc-receptors.False positive report of CDC crossmatch can be caused by the underlying autoimmune diseases such as lupus erythematosus,that may lead to inadvertent refusal of adequate kidney grafts.Detailed study of DSA by molecular technique would prevent wrong exclusion of such donors.Based on these investigations this patient is deemed to have "standard immunological risk" for renal transplantation.

Synchrotron infrared spectral regions as signatures for foodborne bacterial typing

Fourier-transform infrared(FTIR) spectroscopy has emerged as a viable alternative to biochemical and molecular biology techniques for bacterial typing with advantages such as short analysis time, low cost and laboratorial simplicity. In this study, synchrotron radiationbased FTIR(SR-FTIR) spectroscopy with higher spectral quality was successfully applied to type 16 foodborne pathogenic bacterial strains. Combined with principal component analysis(PCA) and hierarchical cluster analysis(HCA), we found that the specific spectral region1300-1000 cm^(-1), which reflects the information of phosphate compounds and polysaccharides, can be used as the signature region to cluster the strains into groups similar with genetic taxonomic method. These findings demonstrated that FTIR spectra combined with HCA have a great potential in quickly typing bacteria depending on their biochemical signatures.

Overview of Molecular Typing Tools for The Characterization of Salmonella enterica in Malaysia

Salmonella is a member of the family Enterobacteriaceae. This genus comprises two species, namely Salmonella enterica and Salmonella bongori. Salmonella enterica is further divided into six subspecies, namely enterica, salamae, arizonae,

DNA TYPING SYSTEM FOR HLA-A2 ALLELES BY POLYMERASE CHAIN REACTION WITH SEQUENCE- SPECIFIC PRIMERS

Objective. To establish a PCR- SSP method for discriminating as many HLA- A* 02 alleles, which could easily be introduced into a routine laboratory. Methods. In this study we typed HLA- A* 02 polymorphisms by a sequence- specific primer (SSP) method, which involved round 1 and round 2 PCR reactions to detect 17 HLA- A* 02 alleles (they are HLA- A* 0201- 0217 alleles) covering exon 2 and exon 3. Results. We have found that DNA sample concentration and purity were the most important variables in determining the quality of the results. For identifying correct band size, the size marker used was important. We noticed that different PCR machines performed differently. By this method, we detected 20 HLA- A* 02 positive genomic DNA samples and found 4 kinds of HLA- A* 02 alleles. They were HLA- A* 0201, 0203, 0206 and 0210. Conclusion. The HLA- A* 02 PCR- SSP method was proven to be a reliable and easily applicable typing method. Our results suggest that the SSP described here provides an optimal HLA- A* 02 typing technique that may be useful in selecting donor- recipient pairs in bone marrow transplantation between unrelated individuals.

Specific primers design based on the superoxide dismutase b gene for Trypanosoma cruzi as a screening tool:Validation method using strains from Colombia classified according to their discrete typing unit

Objective:To classify 21 new isolates of Trypanosoma cruai(T.cruzi) according to the Discrete Typing Unit(DTU) which they belong to,as well as tune up a new pair of primers designed to detect the parasite in biological samples.Methods:Strains were isolated,DNA extracted,and classified by using three Polymerase Chain Reactions(PCR).Subsequently this DNA was used along with other isolates of various biological samples,for a new PCR using primers designed.Finally,the amplified fragments were sequenced.Results:It was observed the predominance of DTU i in Colombia,as well as the specificity of our primers for detection of T.cruzi,while no band was obtained when other species were used.Conclusions:This work reveals the genetic variability of 21 new isolates of T.cruzi in Colombia.Our primers confirmed their specificity for detecting the presence of T.cruzi.

Implementation of a Petrographical and Petrophysical Workflow Protocol for Studying the Impact of Heterogeneity on the Rock Typing and Reservoir Quality of Reefal Limestone:A Case Study on the Nullipore Carbonates in the Gulf of Suez

This study focuses on the heterogeneity of the middle Miocene syn-rift Belayim nullipore(reefal)marine sequences in the Gulf of Suez and its impacts on reservoir quality.The sequences consist of coralline algal reef limestones with a highly complex dual-porosity system of primary and secondary porosities of widely varying percentages.To achieve a precise mathematical modeling of these reservoir sequences,a workflow protocol was applied to separate these sequences into a number of hydraulic flow units(HFUs)and reservoir rock types(RRTs).This has been achieved by conducting a conventional core analysis on the nullipore marine sequence.To illustrate the heterogeneity of the nullipore reservoir,the Dykstra-Parsons coefficient(V)has been estimated(V=0.91),indicating an extremely heterogeneous reservoir.A slight to high anisotropy(λ_(k))has been assigned for the studied nullipore sequences.A stratigraphic modified Lorenz plot(SMLP)was applied to define the optimum number of HFUs and barriers/baffles in each of the studied wells.Integrating the permeability-porosity,reservoir quality index-normalized porosity index(RQI-NPI)and the RQI-flow zone indicator(RQIFZI)plots,the discrete rock types(DRT)and the R35 techniques enable the discrimination of the reservoir sequences into 4 RRTs/HFUs.The RRT4 packstone samples are characterized by the best reservoir properties(moderate permeability anisotropy,with a good-to-fair reservoir quality index),whereas the RRT1 mudstone samples have the lowest flow and storage capacities,as well as the tightest reservoir quality.

Characterization of Xanthomonas citri pv.citri from China based on spoligotyping

Xanthomonas citri pv.citri(Xcc),a gram-negative bacterium,is the causal agent of citrus canker,one of the most devastating diseases threatening the citrus industry worldwide.Understanding the diversity and population structure of Xcc is a prerequisite for disease epidemiological monitoring and effective disease management.Recent characterization of the clustered regularly interspaced short palindromic repeats(CRISPR)/cas(CRISPR-associated proteins genes)system with a highly variable repeat number among species provides a new molecular typing method for bacterial genetic analysis.In this study,we performed systematic in silico analyses of 28 Xcc genomes and identified a credible CRISPR/cas in Xcc strains.Further analysis of CRISPR polymorphisms(repeat number and spacer types)in 129 Xcc A strains collected from six provinces in China identified 15 types of CRISPR arrays with 25 spacers.Phylogenetic analysis of Xcc strains based on the CRISPR locus produced a more reliable and accurate typing result compared to the commonly used loci.In addition,seven associated cas genes—cas1,cas2,cas3,cas4,cas5,cas7(csd2),and cas8(csd1)—were found located adjacent to the CRISPR array.BLAST results showed>99%similarity of seven cas genes among Xcc strains.Homology analysis of spacer sequences showed that six spacers had possible phage/prophage origin.The characterization of the CRISPR/cas system among Xcc strains provided an updated strain typing method for Xcc diversity analysis and yielded a panoramic view of CRISPR evolution for further studies of Xcc-phage interactions.

Identification of Variable-Number Tandem-Repeat(VNTR)Sequences in Acinetobacter pittii and Development of an Optimized Multiple-Locus VNTR Analysis Typing Scheme

Objective To develop a multiple-locus variable-number tandem-repeat(VNTR)analysis(MLVA)assay for Acinetobacter pittii typing.Methods Polymorphic VNTRs were searched by Tandem Repeats Finder.The distribution and polymorphism of each VNTR locus were analyzed in all the A.pittii genomes deposited in the NCBI genome database by BLAST and were evaluated with a collection of 20 well-characterized clinical A.pittii strains and one reference strain.The MLVA assay was compared with pulsed-field gel electrophoresis(PFGE)for discriminating A.pittii isolates.Results Ten VNTR loci were identified upon bioinformatic screening of A.pittii genomes,but only five of them showed full amplifiability and good polymorphism.Therefore,an MLVA assay composed of five VNTR loci was developed.The typeability,reproducibility,stability,discriminatory power,and epidemiological concordance were excellent.Compared with PFGE,the new optimized MLVA typing scheme provided the same and even greater discrimination.Conclusion Compared with PFGE,MLVA typing is a faster and more standardized alternative for studying the genetic relatedness of A.pittii isolates in disease surveillance and outbreak investigation.

Tree Automaton Based Algorithms for Sub-Typing and inDOM Relations in XQuery

Proposed by W3C (World Wide Web Consortium), XQuery is a descriptive query language for XML structured data or documents. The specification of XQuery gives functional description of XQuery’s typing system, but doestn’t provide methods to judge the Sub-typing and inDOM relations in language compiling. In this paper, both of the two important relations: Sub-typing and inDOM, are described and analyzed. After some definitions of Tree Automaton, the judgment algorithms of these two relations are proposed. Key words XQuery - typing system - tree automata CLC number TP 301. 6 Biography: XIE Rong-chuan (1946-), male, Associate professor, research direction: database, internet information system.

Molecular Typing of Aeromonas hydrophila Isolated from Common Carp in Northeast China

A total of 59 isolates of Aeromonas hydrophila were collected from common carp suffering from freshwater fish hemorrhage disease in 13 fishing grounds in the northeast China, and their phenotypic and genetic characteristics were investigated. All of the isolates were identified as A. hydrophila by traditional biochemical method and yielded a 686-bp DNA fragment of the 16S rDNA gene in the PCR experiments. Collected strains were also evaluated for their susceptibility to 17 different antibiotics. The isolates showed an even trend of the resistance and sensitivity to drugs, highly sensitive to antibiotics, such as Levofloxacin, PolymyxinB, Ofloxacin and resistant to antibiotics, such as Bristopen, Lincomycin, Ampicillin, Teicoplanin. Evaluation of genetic diversity was performed on all isolates by molecular typing with enterobacterial repetitive intergenic consensus （ERIC-PCR） method. The results showed that three different types, i.e. type I, ]I, and type ~I, were found in 59 isolates and type III accounted for a large proportion of 54.84%. There was no dominant difference between the tendency of the isolates of Heilongjiang Province and Jilin Province in these three types, which showed DI 〉 I 〉 I, while the isolates of Liaoning Province showed III 〉 II 〉 I. The percentage of different types in different provinces varied in each other; however, they didn＇t show any obvious regional or cluster-specific branches. In conclusion, the ability to distinguish Aeromonas hydrophila strains from diseased common carp with ERIC-PCR would be useful for epidemiological investigation and population genetic analysis of this pathogen in China.

RAPID HLA-DRB1 GENERIC TYPING BY PCR-SSP METHOD

We report a simple and rapid HLA-DRBI generic typing method, PCRSSP, which is practical and inexpensive. We use 9 sequence-specific primers and 2 group specific Primers to define the HI-ADRB1 specificities DR1,DR2, DR3, DR4,DR5,DR6, DR7, DR8, DR9 and DR10. The HLA DR3, DR5,DR6 and DR8 can be amplified by the two primers of DR3568 and DRB1. The DR6 specificity can be identified by excluding the DR3, DR5 and DR8 when the DR3568 are positive. Any individuals can be typed with some exception: the three pairs of phenotype DR3/DR3 and DR3/DR6, DR5/DRS and DR5/DR6,DR8/DR8 and DR8/DR6 cannot be discriminated from each other. We typed 106 unrelated healthy people from Beijing locations in two weeks. We think this typing method is suitable to replace the error-prone serologic HLA-DR tests in routine clinical practice, including the Prospective typing of cadaveric organ donors.

Weather Typing and Dissipation Forecast of Fog in Haizhou Bay

[ Objective] The research aimed to study weather typing and dissipation forecast of the fog in Haizhou Bay. [ Method] Based on the me- teorological observation data of three representative stations in Lianyungang, we analyzed weather situation before fog occurrence as well as the meteorological elements of coastal fog in Haizhou Bay, and established dissipation rating forecast equation of the fog. [ Result] From the surface weather chart, the fog in Haizhou Bay was divided into four types： low-pressure inverted trough type, prefrontal warm-zone type, high-pressure rear type and high-pressure bottom type. FOg formation was closely related to stratification stability, temperature, relative humidity, wind direction and wind velocity. By using multiple linear regression method, dissipation rating prediction equation of the fog was established. Via test, prediction was correct basically, and it reached 77% that forecast rating error was below level 0.5.[Conclusion] The research could provide favorable reference for forecast and warninq of the fo_q in Haizhou Bay.

EXPERIENCE ON HLA TYPING FOR ALLOGENEIC MARROW TRANSPLANTATION IN 217 MEMBERS OF 30 FAMILIES

From April 1978 to Oct 1990, 217 members of 30 families who requiredallogeneic marrow transplantation were examined for HLA typing, with HLA standardantiserum and mixed lymphocyte culture (MLC). The following conclusions could bedrawn: (1) when the HLA-A, B, C loci between donor and recipient were identical, theD locus also was generally identical too, but, in some rare cases the HLA-A, B, C wereidentical while the D locus remained different detected by MLC SI which wassignificantly increased; (2) in addition to twin, the sib were the main candidates of donorsin allogeneic marrow transplantation, however, if the phenotype of children was compati-ble to that of their parents with no significant stimulation in MLC, the parents could alsobe considered as a donor; (3) if the HLA-A, B, C typing was identical between fatherand motaher, the parents can also be considered as an eligible donor. Using HLA-A, B,C, DR and D loci identical sibs as donor, 9 of 11 cases (aplastic anemia 9 cases andANLL-M2 2 cases who received allogeneic marrow transplantation) had the evidence ofengraftment. Much attention should be paid to the difference of the minorhistocompatibility antigens, such as sex associated antigen between donor and recipient, be-cause even in 11 cases with HLA-A, B, C, DR and D loci identical sibs as donors formarrow transplantation, 5 cases still had the manifestation of GVHD in various degree,4/5 died of severe GVHD.

APPLICATION OF DIGOXIGENIN LABELED PROBES TO DETECTION AND TYPING OF HERPES SIMPLEX VIRUS

A total of 44 HSV isolates from the patients was detected and typed by using HSV typespecific DNA probes labeled with Digoxigenin and 32 P respectively. 25 isolates were identified to be HSV-1, 19 were HSV-2. Comparison of the typing results showed that the sensitivity and specificity of the Digoxigenin-labeled probe were equal to those of the 32 P-labeled Probe, and the typing rate of HSV isolates was 100%. The Dig-labeled probes are nonradioactive and the labeling technigue is simple, the Dig-labeled probes can be stored and reused, so they are more valuable for clinical application.

Molecular Typing of Clinical and Environmental <i>Cryptococcus neoformans</i>Strains Isolated in Italy

Cryptococcus neoformans is an encapsulated yeast causing mainly opportunistic infections. DNA molecular typing techniques divided C. neoformans into four major molecular types (AFLP1/VNI, AFLP1A/VNB/VNII, AFLP1B/ VNII, AFLP3/VNIII, AFLP2/VNIV) characterized by different pathogenicity, geographical distribution and susceptibility to antifungal treatments. In this study 170 Italian C. neoformans clinical isolates (CI) and 32 environmental isolates (EI), collected and serotyped during a ten-year period (1985 to 1995), were genotyped using [GACA]4 microsatellite PCR fingerprinting. The molecular types were compared to their geographic distribution, specimen sources and patient’s risk factors. All four molecular types were described among the CI and only VNI and VNIV among the EI. VNIV molecular type was isolated with significant prevalence among the CI and VNI among the EI. A different geographical distribution of molecular types was detected: VNIV was the most prevalent in the North and Center of Italy;VNIII was isolated almost exclusively in the Center. No significant correlation among molecular types versus predisposing diseases or isolation sources was detected. The strains isolated from different body sites of the same patient (17 cases) were of the same genotype. Five out 9 cases relapsed with a different molecular type. This preliminary investigation shows a high intraspecies variability and reveals a nonhomogeneous distribution of C. neoformans molecular types in Italy.