Detection Of Antimicrobial Resistance Genes Research Articles

Molecular Characteristics of Methicillin-Resistant Staphylococci Clinical Isolates from a Tertiary Hospital in Northern Thailand.

Methicillin-resistant staphylococci are now recognized as a major cause of infectious diseases, particularly in hospitals. Molecular epidemiology is important for prevention and control of infection, but little information is available regarding staphylococcal infections in Northern Thailand. In the present study, we examined antimicrobial susceptibility patterns, detection of antimicrobial resistance genes, and SCCmec types of methicillin-resistant S. aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) isolated from patients in a hospital in Northern Thailand. The species of MRSA and MR-CoNS were identified using combination methods, including PCR, MALDI-TOF-MS, and tuf gene sequencing. The susceptibility pattern of all isolates was determined by the disk diffusion method. Antimicrobial resistance genes, SCCmec types, and ST239 were characterized using single and multiplex PCR. ST239 was predominant in MRSA isolates (10/23). All MR-CoNS (N=31) were identified as S. haemolyticus (N=18), S. epidermidis (N=3), S. cohnii (N=3), S. capitis (N=6), and S. hominis (N=1). More than 70% of MRSA and MR-CoNS were resistant to cefoxitin, penicillin, oxacillin, erythromycin, clindamycin, gentamicin, and ciprofloxacin. In MRSA isolates, the prevalence of ermA (78.3%) and ermB (73.9%) genes was high compared to that of the ermC gene (4.3%). In contrast, ermC (87.1%) and qacA/B genes (70.9%) were predominant in MR-CoNS isolates. SCCmec type III was the dominant type of MRSA (13/23), whereas SCCmec type II was more present in S. haemolyticus (10/18). Ten MRSA isolates with SCCmec type III were ST239, which is the common type of MRSA in Asia. This finding provides useful information for a preventive health strategy directed against methicillin-resistant staphylococcal infections.

Characterization of virulence factors, antimicrobial resistance patterns and biofilm formation of Pseudomonas aeruginosa and Staphylococcus spp. strains isolated from corneal infection

Infectious keratitis is a serious ocular infection that can lead to severe visual impairment and blindness. Bacterial pathogens are responsible for nearly half of infectious keratitis cases. This study was performed to determine the virulence factors, antimicrobial resistance patterns, and biofilm formation ability of Pseudomonas aeruginosa and Staphylococcus spp. strains isolated from corneal infections. A total of 56 corneal scraping samples were collected over 8 months. P.aeruginosa and staphylococcal strains were identified by phenotypic and genotypic methods. Determination of multidrug resistance was performed according to its definition of multidrug resistance (MDR). Detection of antimicrobial resistance genes and determinants of virulence were also performed using standard procedures. Biofilm formation ability of the isolates was determined by colorimetric microtitration plate assay and Modified Congo red agar (MCRA). In the present study, P.aeruginosa, MSSA, MRSA, MS-CoNS and MR-CoNS strains were isolated from corneal infections. Multidrug resistance was observed in 42.9% and 57.1% of P.aeruginosa and Staphylococcus spp., respectively. The most frequent virulence genes among P.aeruginosa strains were exoA and exoS (100%) followed by exoU (71.4%) and lasB (28.6%). All the P.aeruginosa isolates were biofilm producers and carried the algD gene (100%). All staphylococcal strains were negative for pvl gene amplification. Biofilm formation was also observed in 4 (57.1%) isolates. Both icaA and icaD genes were detected in the biofilm producers. Our results indicated that P.aeruginosa and Staphylococcus spp. were the most prevalent bacterial agents that cause corneal infections. However, their virulence traits and biofilm formation ability were noteworthy.

Antimicrobial resistance genes in pathogenic Escherichia coli isolated from diseased broiler chickens in Egypt and their relationship with the phenotypic resistance characteristics.

Aim:The aim of this study was to determine the relationship between phenotypic resistance and genotypic resistance of isolated serotyped pathogenic Escherichia coli isolates from the clinically diseased broiler.Materials and Methods:A total of 160 samples (heart, liver, kidney, and lung) were collected from 18 to 34 days old clinically diseased broiler from 40 broiler farms (3-5 birds/farm) reared in Giza and Kaluobaia Governorates for the isolation of pathogenic E. coli. Various E. coli isolates were tested for the pathogenicity based on Congo red (CR) dye binding assay. The obtained CR-positive E. coli isolates were subjected to serological identification using slide agglutination test. Disc diffusion test was used to study the sensitivity pattern of E. coli isolates to available 12 antibiotics. Polymerase chain reaction was performed for the detection of antimicrobial resistance genes in the studied pathogenic E. coli isolates.Results:The results revealed that 56 samples (35 %) were positive for E. coli. The results of the CR assay indicates that 20 isolates of 56 (35.7%) were positive and 36 isolates (64.3%) were negative. Identified E. coli serotypes of CR-positive isolates were 1 (O24), 2 (O44), 2 (O55), 5 (O78), 2 (O86), 1 (124), 3 (O127), 1 (O158), and 3 untyped. Resistance rate in disc diffusion test was 85% to oxytetracycline and kanamycin; 80% to ampicillin (AMP), clindamycin, and streptomycin (S); 75% to enrofloxacin; 65% to chloramphenicol; 55% to cefotaxime and gentamicin (CN); 45% to trimethoprim+sulfamethoxazole; 35% to erythromycin (ERI); and 30% to oxacillin. All strains are multidrug-resistant (MDR). Antibacterial resistance genes CITM, ere, aac (3)-(IV), tet(A), tet(B), dfr(A1), and aad(A1) were detected in 14 (70%), 12 (60%), 12 (60%), 8 (40%), 11 (55%), 8 (40%), and 9 (45%) of tested 20 isolates, respectively. Multidrug resistance was detected in the form of resistance to 42%-83.3% of tested 12 antibiotics. Three isolates (15%) of 20 tested isolates showed a relationship between phenotype and genotype and 17 (85%) showed irregular relation. Strains are sensitive and show resistant gene (P-G+) presented in three isolates for AMP (beta-lactam), one for ERI (Macrolide), as well as five isolates for trimethoprim (pyrimidine inhibitor). E. coli isolates had resistance and lacked gene (P+ G-) reported meanly in one isolate for CN (aminoglycoside), two isolates for tetracycline, four isolates for ERI, seven isolates for trimethoprim, and eight isolates for S (aminoglycoside).Conclusion:The study demonstrates that E. coli is still a major pathogen responsible for disease conditions in broiler. E. coli isolates are pathogenic and MDR. Responsible gene was detected for six antibiotics in most of the isolates, but some do not show gene expression, this may be due to few numbers of resistance genes tested or other resistance factors not included in this study.

Fast and sensitive isothermal DNA assay using microbead dielectrophoresis for detection of anti-microbial resistance genes

Antimicrobial resistant pathogens are a growing worldwide threat to human health. This study describes a novel method for rapid and sensitive detection of antimicrobial resistance (AMR) genes, specifically blaCTX-M-15 which encodes for the enzyme that offers resistance to extended spectrum β-lactam antibiotics. The method combines isothermal DNA amplification by recombinase polymerase amplification (RPA), with microbead dielectrophoresis (DEP)-based DNA detection. The RPA amplicon is captured onto dielectric microbeads, and the amount of amplicon determined by dielectrophoretic impedance measurement (DEPIM) of the microbeads. Amplicon-labeled microbeads were prepared by either a two-step or one-step method. A purified recombinant plasmid containing blaCTX-M-15 and genomic DNA (with plasmid) extracted from an AMR bacteria (Escherichia coli NCTC 13441) were used as target samples. A one-step method in which RPA and DNA immobilization on the microbeads is carried out simultaneously, has a detection limit of 2 copies/reaction for pure plasmid and 50 copies/reaction for genomic DNA. The assays are quantitative with a dynamic range up to 105 copies/reaction, with a total detection time of 26 min. Both methods are easy, rapid, and unlike lateral flow detection are quantitative.

Detection of antimicrobial resistance genes of Helicobacter pylori strains to clarithromycin, metronidazole, amoxicillin and tetracycline among Egyptian patients

BackgroundAntibiotic resistance of Helicobacter pylori (H. pylori) treatment is on the rise, and is affecting the efficacy of current used therapeutic regimens. AimWe aimed to enhance the understanding of antimicrobial resistance rates of H. pylori strains recovered from patients at Theodor Bilharz Research Institute Hospital in Egypt, as a mandatory step before starting treatment. Subjects and methodsMutant genes conferring metronidazole, amoxicillin, clarithromycin, and tetracycline resistance were detected in 60 H. pylori strains recovered from patients who underwent upper endoscopic examination. Patients were considered to be infected with H. pylori when rapid urease test and detection of 16S rRNA in gastric biopsies recorded positive. Molecular detection of resistant genes to metronidazole (rdx gene) and amoxicillin (pbp1A gene) was carried out by conventional PCR followed by sequencing of PCR products. While detection of 23S rRNA gene conferring clarithromycin resistance and detection of 16S rRNA mutation gene conferring tetracycline resistance were carried out by real-time PCR. ResultsH. pylori resistance rates to metronidazole, and amoxicillin were 25% and 18.3% respectively. While for clarithromycin and tetracycline, point mutations in 23S rRNA types A2142G and A2143G and in 16S rRNA of H. pylori were assessed by real time PCR assay respectively. Resistance mutant genes were found to be 6.7% of clarithromycin and 1.7% of tetracycline. Combined resistance rates to metronidazole and amoxicillin was (11.6%) followed by metronidazole and clarithromycin (5%), while patterns of clarithromycin and amoxicillin (1.6%), metronidazole, clarithromycin and amoxicillin (1.6%) were revealed. ConclusionData concerning antimicrobial resistance genes play an important role in empiric treatment of H. pylori infection. According to our results, H. pylori resistance to metronidazole and amoxicillin was relatively high. Clarithromycin is still a good option for first line anti-H. pylori treatment. Combined resistant strains are emerging and may have an effect on the combination therapy.

Evaluation of Sepsis Flow Chip for identification of Gram-negative bacilli and detection of antimicrobial resistance genes directly from positive blood cultures

Blood stream infections are serious conditions associated with high morbi-mortality. In this study, the new Sepsis Flow Chip (SFC) assay for identification of Gram-negative bacteria and their antimicrobial resistance genes was evaluated in positive blood cultures (BCs). SFC is a microarray with a broad panel comprising the most frequent causative agents of sepsis and antimicrobial resistance genes associated with them. A total of 100 prospective BCs, positive for Gram-negative bacilli, were assessed in the routine of the clinical microbiology laboratory and also applying the SFC assay. Moreover, 19 BCs spiked with well-characterized enterobacterial isolates, harboring antimicrobial resistance genes, were analyzed by the latter. Among the monomicrobial BCs (90), the concordance between SFC identification and the reference method was 94.4%; however, it achieved 100% when SFC was combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after 4-h incubation. Regarding polymicrobial BCs (10), 15 out of the 22 bacteria present (68.2%) were correctly identified, including all contained in 50% of the cultures. With regard to antimicrobial resistance genes, 98.8%, 98.9%, and 99% concordance was obtained for blaCTX-M, blaOXA-48, and blaVIM, respectively, in comparison with polymerase chain reaction amplification. SFC assay gives results in only 4 h and showed a high concordance rate with the reference method. Although further evaluation studies are necessary, SFC assay implementation, together with antimicrobial stewardship programs, could contribute to improve the therapeutic approaches and to reduce the morbi-mortality, length of hospital stay, and healthcare-associated costs in patients with sepsis.

Detection of antimicrobial resistance genes associated with the International Space Station environmental surfaces

Antimicrobial resistance (AMR) is a global health issue. In an effort to minimize this threat to astronauts, who may be immunocompromised and thus at a greater risk of infection from antimicrobial resistant pathogens, a comprehensive study of the ISS “resistome’ was conducted. Using whole genome sequencing (WGS) and disc diffusion antibiotic resistance assays, 9 biosafety level 2 organisms isolated from the ISS were assessed for their antibiotic resistance. Molecular analysis of AMR genes from 24 surface samples collected from the ISS during 3 different sampling events over a span of a year were analyzed with Ion AmpliSeq™ and metagenomics. Disc diffusion assays showed that Enterobacter bugandensis strains were resistant to all 9 antibiotics tested and Staphylococcus haemolyticus being resistant to none. Ion AmpliSeq™ revealed that 123 AMR genes were found, with those responsible for beta-lactam and trimethoprim resistance being the most abundant and widespread. Using a variety of methods, the genes involved in antimicrobial resistance have been examined for the first time from the ISS. This information could lead to mitigation strategies to maintain astronaut health during long duration space missions when return to Earth for treatment is not possible.

Molecular Methods for Detection of Antimicrobial Resistance.

The increase in bacteria harboring antimicrobial resistance (AMR) is a global problem because there is a paucity of antibiotics available to treat multidrug-resistant bacterial infections in humans and animals. Detection of AMR present in bacteria that may pose a threat to veterinary and public health is routinely performed using standardized phenotypic methods. Molecular methods are often used in addition to phenotypic methods but are set to replace them in many laboratories due to the greater speed and accuracy they provide in detecting the underlying genetic mechanism(s) for AMR. In this article we describe some of the common molecular methods currently used for detection of AMR genes. These include PCR, DNA microarray, whole-genome sequencing and metagenomics, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The strengths and weaknesses of these methods are discussed, especially in the context of implementing them for routine surveillance activities on a global scale for mitigating the risk posed by AMR worldwide. Based on current popularity and ease of use, PCR and single-isolate whole-genome sequencing seem irreplaceable.

Antimicrobial resistance and genetic diversity of the SXT element in Vibrio cholerae from clinical and environmental water samples in northeastern Thailand

Multidrug resistance in V. cholerae has been increasing around the world including northeastern Thailand. The aquatic environment is a reservoir of V. cholerae and might be an important source of resistant strains. The aims of this study were to investigate the phylogenetic relationships of intSXT gene sequences from 31 clinical and 14 environmental V. cholerae O1 and non-O1/non-O139 isolates and 11 sequences amplified directly from environmental water samples. We also amplified class 1 integrons, the SXT elements (targeting the intSXT gene) and antimicrobial resistance genes directly from water samples. Phylogenetic analysis displayed two major distinct clusters (clusters 1 and 2). Most V. cholerae O1 (19/20, 95%) and non-O1/non-O139 isolates (8/11, 72.7%) from clinical sources, and all sequences obtained directly from water samples, belonged to cluster 1. Cluster 2 mostly comprised environmental non-O1/non-O139 isolates (10/12, 83.3%). We successfully amplified the SXT elements directly from17.5% of water samples. Associated resistance genes were also amplified as follows: sul2 (41.3% of water samples), dfrA1 (60%), dfr18 (33.8%), strB (70%) and tetA (2.5%). Class 1 integrons were not found in water samples, indicating that the SXT element was the major contributor of multidrug resistance determinants in this region. The SXT element and antimicrobial resistance genes could be transferred from clinical V. cholerae O1 to environmental V. cholerae non-O1/non-O139 was demonstrated by conjugation experiment. These findings indicate that there may have been cross dissemination and horizontal gene transfer (HGT) of the SXT element harbored by V. cholerae O1 and non-O1/non-O139 strains isolated from clinical and environmental water sources. Environmental water might be an important source of antimicrobial resistance genes in V. cholerae in this region. Direct detection of antimicrobial resistance genes in water samples can be used for monitoring the spread of such genes in the ecosystem.

Clinical relevance of molecular identification of microorganisms and detection of antimicrobial resistance genes in bloodstream infections of paediatric cancer patients.

BackgroundBloodstream infections (BSIs) are the major cause of mortality in cancer patients. Molecular techniques are used for rapid diagnosis of BSI, allowing early therapy and improving survival. We aimed to establish whether real-time quantitative polymerase chain reaction (qPCR) could improve early diagnosis and therapy in paediatric cancer patients, and describe the predominant pathogens of BSI and their antimicrobial susceptibility.MethodsBlood samples were processed by the BACTEC system and microbial identification and susceptibility tests were performed by the Phoenix system. All samples were screened by multiplex 16 s rDNA qPCR. Seventeen species were evaluated using sex-specific TaqMan probes and resistance genes blaSHV, blaTEM, blaCTX, blaKPC, blaIMP, blaSPM, blaVIM, vanA, vanB and mecA were screened by SYBR Green reactions. Therapeutic efficacy was evaluated at the time of positive blood culture and at final phenotypic identification and antimicrobial susceptibility results.ResultsWe analyzed 69 episodes of BSI from 64 patients. Gram-positive bacteria were identified in 61 % of the samples, Gram-negative bacteria in 32 % and fungi in 7 %. There was 78.2 % of agreement between the phenotypic and molecular methods in final species identification. The mecA gene was detected in 81.4 % of Staphylococcus spp., and 91.6 % were concordant with the phenotypic method. Detection of vanA gene was 100 % concordant. The concordance for Gram-negative susceptibilities was 71.4 % for Enterobacteriaceae and 50 % for Pseudomonas aeruginosa. Therapy was more frequently inadequate in patients who died, and the molecular test was concordant with the phenotypic susceptibility test in 50 %.ConclusionsqPCR has potential indication for early identification of pathogens and antimicrobial resistance genes from BSI in paediatric cancer patients and may improve antimicrobial therapy.

Clinical Relevance of Molecular Microbiology

Molecular methods for the detection and characterization of microorganisms have transformed diagnostic microbiology. Detection of microorganisms was previously laborious and time taking by conventional microbiological methods but now more rapid detection by molecular methods is possible for pathogens of public health importance. Detection of antimicrobial resistance genes and their characterization by genotyping is also feasible. Detection of viral resistance gene and testing of viral load for the monitoring of antiviral therapies are possible because of molecular technique and automation of molecular microbiology. This review will focus on basic molecular techniques and the clinical utility of these molecular methods in the management of infectious diseases.

Detection of antimicrobial resistance genes in Diarrhoeagenic Escherichia coli from children less than 5 years

Detection of antimicrobial resistance genes in Diarrhoeagenic Escherichia coli from children less than 5 years

Detection of antimicrobial resistance genes in betalactamase- and carbapenemase-producing Klebsiella pneumoniae by patient surveillance cultures at an intensive care unit in Rio de Janeiro, Brazil

ABSTRACT Introduction: The increasing incidence of multi-resistant microorganisms has been considered a public health problem. One of the routines included in hospital practice is the screening of colonized and/or infected patients. Objective: The aim of this study was to evaluate the genetic variability and clonal relationships of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae, from surveillance cultures, at an intensive care unit, in Rio de Janeiro, Brazil. Material and methods: Seventy K. pneumoniae isolates were obtained from rectal swabs (March 2013 to March 2014). Antimicrobial susceptibility was assessed by VITEK 2 System. Resistant genes blaSHV, blaTEM, blaOXA-1, blaKPC, blaOXA-48, blaCTX-M-15, blaVIM, blaIMP and blaNDM were investigated by polymerase chain reaction (PCR); genetic diversity, by Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). Results: Strains showed high resistance rates to cefepime (94%), ceftazidime (96%), ertapenem (61%), imipenem (54%) meropenem (43%) and ciprofloxacin (69%). The most prevalent genes were blaSHV (69%), blaTEM (63%), blaOXA-1 (60%), blaKPC (57%), blaCTX-M-15 (47%), blaOXA-48 (16%). Genes blaVIM, blaIMP and blaNDM were not detected. Twenty nine profiles of resistance genes were observed, with 23% carrying at least five genes. A great genetic diversity (68 ERIC profiles) was also observed among the strains. Conclusion: Although no clonal relationship was observed within the isolates, this study revealed alarming data on the antimicrobial resistance deficiently monitored for preventive purposes in Brazil. Our data allow us to conclude that the inclusion of surveillance cultures in health facilities is a recommended strategy aiming particularly at preventing the spread of resistance genes in the hospital environment and, consequently, reducing morbidity and mortality.

Boiling, Freezing - Thawing and Simple Chemical Extraction Methods Are Comparable to Costly Kit - Based Bacterial DNA Extraction Methods as Evidenced by Conventional and Real-Time PCR

Background: Bacterial DNA extraction is a preliminary step for PCR studies carried out on bacteria for diagnosis and detection of antimicrobial resistance genes. Objectives: Testing efficiency of two physical methods (boiling and freezing-thawing) and two chemical methods (SDS and tween based methods) for extraction of bacterial DNA suitable for conventional as well as real time PCRs in comparison to a costly ready to use kit method. Methodology: Escherichia coli ATCC 25922 was submitted for five DNA extraction methods including QIAamp ready kit, boiling, freezing-thawing in TE, SDS method, and tween method. DNA purification by ethanol precipitation and wash was carried out after tested physical and chemical methods. The extracted DNAs were assessed by Nanodrop spectrophotometer, conventional PCR followed by Uviband quantification, and semiquantitative SYBR green based RT- PCR. Results: Nanodrop assessment showed that QIAamp kit and tween method produced the highest DNA yields; 78.1 ng/µl and 49.72 ng/µl, respectively, while SDS method gave the lowest. Both QIAamp kit and tween methods produced comparable DNA in concentration and purity. All tested methods extracted DNA amplifiable by PCR. In conventional PCR, the concentrations ranged from 287.31 to330.46 ng/5µl for boiling and QIAamp kit, respectively. In RT-PCR, the highest CT values were obtained by OIAmp kit, followed by tween, freezing-thawing in TE, SDS, and boiling methods with narrow range of variation (from 30.80 to 31.85). No significant statistical differences were detected between tested methods in the results of PCRs either conventional or real time. Conclusion: Boiling in water, freezing-thawing in TE, tween and SDS based methods followed by DNA precipitation provide rapid, effective, less laborious and truly cost-effective methods for extraction of bacterial DNA suitable for PCR studies.

Early insights into the potential of the Oxford Nanopore MinION for the detection of antimicrobial resistance genes.

ObjectivesGenome sequencing will be increasingly used in the clinical setting to tailor antimicrobial prescribing and inform infection control outbreaks. A recent technological innovation that could reduce the delay between pathogen sampling and data generation is single molecule sequencing. An example of this technology, which is undergoing evaluation through an early access programme, is the Oxford Nanopore MinION.MethodsWe undertook a feasibility study on six clinically significant pathogens, comparing the MinION to the Illumina MiSeq and PacBio RSII platforms. Genomic DNA was prepared and sequenced using the MinION as instructed by the manufacturer, and Illumina MiSeq and PacBio sequencing was performed using established methods.ResultsAn evaluation of the accuracy of the MinION based on sequencing of an MRSA isolate showed that error rates were higher in the MinION reads, but provided an even coverage across the entire genome length. The MinION detected all of the expected carbapenemases and ESBL genes in five Gram-negative isolates and the mecA gene in an MRSA isolate.ConclusionsThe MinION can detect the presence of acquired resistance genes, but improvements in accuracy are needed so that antimicrobial resistance associated with mutations in chromosomal genes can be identified.

Search Engine for Antimicrobial Resistance: A Cloud Compatible Pipeline and Web Interface for Rapidly Detecting Antimicrobial Resistance Genes Directly from Sequence Data.

BackgroundAntimicrobial resistance remains a growing and significant concern in human and veterinary medicine. Current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria are limited in their effectiveness and scope. With the rapidly developing field of whole genome sequencing beginning to be utilised in clinical practice, the ability to interrogate sequencing data quickly and easily for the presence of antimicrobial resistance genes will become increasingly important and useful for informing clinical decisions. Additionally, use of such tools will provide insight into the dynamics of antimicrobial resistance genes in metagenomic samples such as those used in environmental monitoring.ResultsHere we present the Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally acquired antimicrobial resistance genes in raw sequencing data. The pipeline provides gene information, abundance estimation and the reconstructed sequence of antimicrobial resistance genes; it also provides web links to additional information on each gene. The pipeline utilises clustering and read mapping to annotate full-length genes relative to a user-defined database. It also uses local alignment of annotated genes to a range of online databases to provide additional information. We demonstrate SEAR’s application in the detection and abundance estimation of antimicrobial resistance genes in two novel environmental metagenomes, 32 human faecal microbiome datasets and 126 clinical isolates of Shigella sonnei.ConclusionsWe have developed a pipeline that contributes to the improved capacity for antimicrobial resistance detection afforded by next generation sequencing technologies, allowing for rapid detection of antimicrobial resistance genes directly from sequencing data. SEAR uses raw sequencing data via an intuitive interface so can be run rapidly without requiring advanced bioinformatic skills or resources. Finally, we show that SEAR is effective in detecting antimicrobial resistance genes in metagenomic and isolate sequencing data from both environmental metagenomes and sequencing data from clinical isolates.

Detection of methicillin-resistant Staphylococcus pseudintermedius ST169 and novel ST354 SCCmec II-III isolates related to the worldwide ST71 clone.

The recent appearance of methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a concern for both veterinary and human healthcare. MRSP clonal lineages with sequence type (ST) 71-spa t02-staphylococcal cassette chromosome mec (SCCmec) II-III and ST68-spa t06-SCCmec V have spread throughout Europe and North America, respectively. The current study compared the molecular characteristics of 43 MRSP isolates from dogs in Japan with those of MRSP from previous reports using multilocus sequence typing based on seven housekeeping genes, SCCmec typing, and detection of antimicrobial resistance genes. Three related clonal lineages, ST71, ST169, and the newly registered ST354, were observed in SCCmec II-III isolates from Japan, despite MRSP SCCmec II-III isolates being thought to belong to a single clonal lineage. The majority of SCCmec II-III isolates belonging to ST169 (9/11) and ST354 (3/3), but not ST71 (0/11), harboured tetM. Four STs were observed for the SCCmec V isolates; however, neither ST68 nor related STs were found in the Japanese MRSP isolates. In conclusion, MRSP SCCmec II-III isolates from Japan belonged to ST71 and related STs (ST169 and ST354). A variety of MRSP SCCmec V clones, including some novel clones, were identified.

Multi-drug resistance in Salmonella serovars of zoonotic importance and detection of anti-microbial resistance genes*

Total 50 Salmonella isolates belonging to 10 different serovars comprising Salmonella Typhimurium (21), S. Weltevreden (12), S. Ughelli (5), S. Essen (3), S. Elisabethville (2), S. Lagos (2), S. Drogana (2), S. Enteritidis (1), S. London (1) and un-typable Salmonella-isolate (1) were isolated from 1,132 different samples. Extreme variation of anti-microbial resistance in Salmonella isolates was observed in a range from 0–100%. Maximum resistance was observed against sulphamethizole and furazolidone (100% each) followed by kanamycin (50%), gentamicin (44%), nalidixic acid (14%), tobramycin (10%), amikacin, ampicillin, streptomycin and tetracycline (8% each), amoxicillin/clavulanic acid (6%), norfloxacin (4%), ciprofloxacin and cefotaxime (2% each) and chloramphenicol (0%). High level of sensitivity in Salmonella isolates was observed against chloramphenicol (100%) and tetracycline (92%). The anti-microbial resistance genes detected were blaTEM, tetA, aphAI-LAB, aadB and strA in Salmonella serovars that showed resistance against ampicillin, tetracycline, kanamycin, gentamicin and streptomycin, respectively. The blaTEM gene was present in all ampicillin resistant Salmonella isolates (100%) and strA gene in all streptomycin resistant Salmonella isolates (100%) followed by tetA gene in 75% tetracycline resistant Salmonella isolates, aadB gene in 14.28% gentamicin resistant Salmonella isolates and aphAI-LAB gene in 8% kanamycin resistant Salmonella isolates. All Salmonella isolates (100%) were multi-drug resistant (MDR), indicating injudicious and extensive use of anti-microbial agents in these areas that may pose great risks to animal health and production. Moreover, MDR Salmonella serovars may pose great public health problems through consumption of foods of animal origin. Multi-drug resistance genes exhibited by all Salmonella serovars also indicated serious threats to the animal health and production including great risks to the public health.

Characterisation of Class 1 Integron among <i>Escherichia coli</i> Isolated from Mansoura University Hospitals in Egypt

Extensive use of antibacterials in clinical practice has been associated with increasing frequency of multi-resistant E. coli strains. Genetic elements such as Class 1 integrons have an important role in resistance development. In the current study, a total of 84 E. coli clinical isolates from Mansoura hospitals patients in Egypt were screened for antibacterial susceptibility against 12 different antibacterials. High resistance rates were identified for Ampicillin (92.9%) and sulfamethoxazole/ trimethoprim (84.5%). Class 1 integron was investigated in E. coli isolates by PCR. As a result, Class 1 integron was identified in 51.2% of these isolates. The contents of amplified integron varriable regions, were digested by Alu I restriction endonuclease. Cluster analysis of Class 1 integron digested varriable regions revealed that RFLP digested fragments generated could be classified into 9 different patterns, namely A, B, C, D, E, F, G, H and I. The most prevalent genotype was identifed in Group D. PCR and sequencing were used for detection of antimicrobial resistance genes harbored in integrons of Group D. As a result, main phylogenetic group identified harbored integron cassette carrying resistance gene for two antimicrobial groups namely aminoglycoside and trimethoprim. Multiresistance profiles in Group D exhibited association between antimicrobial resistance and integron presence. These findings suggest that the strategy for treatment of patients with E. coli infections needs to be revised. Furthermore, the high prevalence of Class 1 integron carrying gene confirms the importance of integron-mediated antimicrobial gene cassettes.

Solid and Suspension Microarrays for Microbial Diagnostics.

Advancements in molecular technologies have provided new platforms that are being increasingly adopted for use in the clinical microbiology laboratory. Among these, microarray methods are particularly well suited for diagnostics as they allow multiplexing, or the ability to test for multiple targets simultaneously from the same specimen. Microarray technologies commonly used for the detection and identification of microbial targets include solid-state microarrays, electronic microarrays and bead suspension microarrays. Microarray methods have been applied to microbial detection, genotyping and antimicrobial resistance gene detection. Microarrays can offer a panel approach to diagnose specific patient presentations, such as respiratory or gastrointestinal infections, and can discriminate isolates by genotype for tracking epidemiology and outbreak investigations. And, as more information has become available on specific genes and pathways involved in antimicrobial resistance, we are beginning to be able to predict susceptibility patterns based on sequence detection for particular organisms. With further advances in automated microarray processing methods and genotype–phenotype prediction algorithms, these tests will become even more useful as an adjunct or replacement for conventional antimicrobial susceptibility testing, allowing for more rapid selection of targeted therapy for infectious diseases.