Molecular identification of CTX-M and blaOXY/K1 β-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons

<p>Abstract</p> <p>Background</p> <p>Plasmid encoded ^<it>bla</it>CTX-M enzymes represent an important sub-group of class A β-lactamases causing the ESBL phenotype which is increasingly found in <it>Enterobacteriaceae </it>including <it>Klebsiella </it>spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment.</p> <p>Methods</p> <p>Multiple displacement amplified DNA derived from 20 <it>K. pneumoniae </it>and 34 <it>K. oxytoca </it>clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of ^<it>bla</it>CTX-M and ^<it>bla</it>OXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer.</p> <p>Results</p> <p>Nine out of 20 <it>K. pneumoniae </it>clinical isolates had a ^<it>bla</it>CTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 <it>K. oxytoca </it>clinical isolates. Molecular identification and differentiation between ^<it>bla</it>CTX-M and ^<it>bla</it>OXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. <it>In silico </it>analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located ^<it>bla</it>OXY and K1-genes in <it>Klebsiella </it>spp. and K1-like genes in other <it>Enterobacteriaceae</it>.</p> <p>Conclusion</p> <p>The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of ^<it>bla</it>CTX-M, and ^<it>bla</it>OXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced.</p

Extended-spectrum beta-lactamase producing Enterobacteriaceae : aspects on detection, epidemiology and multi-drug resistance

Beta-lactam antibiotics are the largest and most commonly used group of antimicrobial agents in Sweden as well as world-wide. They show very good tolerability and many of the drugs can be administrated orally. Bacteria expressing extended-spectrum beta-lactamases (ESBLs), enzymes hydrolysing penicillins and cephalosporins, may not respond to therapy using some of these antibiotics. The isolates are also often co-resistant to other antimicrobial agents, thus further limiting treatment options. Often parenterally administrated carbapenems is one of few safe treatment options left. In this thesis we have investigated the occurrence of ESBL producing Enterobacteriaceae in clinical isolates from Östergötland, Sweden, from 2002 until end of 2007 and the occurrence of multiresistance among ESBL producing E. coli. During these investigations we developed a simple method well suited for high-throughput analysis, for detection and sub typing of common ESBL genes. During the six year period, the prevalence of ESBL producing Enterobacteriaceae in Östergötland was very low, &lt;1%, but increasing. The number of patients with ESBL producing E. coli increased significantly from 5 to 47 per year; K. pneumoniae remained between one and four per year. The genes found were dominated by CTX-M group 1 (67%), followed by group 9 (27%). There has been no reason to suspect an outbreak of nosocomial origin. The total consumption of antimicrobial agents was 10.7-12.1 DID per year in primary care; 1.14-1.30 DID per year in hospital care. Of eight oral agents tested, only three showed a generally high susceptibility; mecillinam (91%), nitrofurantoin (96%) and fosfomycin (99%). The corresponding figures for the fifteen tested parenterally administrated drugs were; amikacin (96%), tigecycline (99%), colistin (99%) and ≥99% susceptibility for the carbapenems. Sixty eight percent of the isolates were multiresistant. The most common multiresistance pattern was ESBL phenotype with decreased susceptibility to trimethoprim, trimethoprimsulfamethoxazole, ciprofloxacin, gentamicin and tobramycin. A significant difference in susceptibility between CTX-M groups, in favor of group 9 over group 1, was seen for many of the antibiotics tested; amoxicillin-clavulanic acid, aztreonam, cafepime, ceftibuten, ceftazidime, ciprofloxacin, gentamicin, piperacillin-tazobactam, temocillin, and tobramycin. In conclusion this thesis shows that the prevalence of ESBL producing Enterobacteriaceae in Östergötland was very low but increasing, and the total consumption of antimicrobial agents was stable. A majority of the isolates were multiresistant and a significant difference in susceptibility between CTX-M groups, in favor of group 9 over group 1, was seen for many of the antimicrobial agents tested

Molecular identification of blaSHV, blaLEN and blaOKP β-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons

Plasmid encoded blaSHV enzymes represent an important sub-group of class A β-lactamases causing an ESBL-phenotype which is increasingly found in Enterobacteriaceae including Klebsiella pneumoniae. The chromosomally encoded β-lactamase blaLEN and blaOKP enzymes, which so far only have been found in K. pneumoniae, do not hydrolyse extended-spectrum cephalosporins. In the present study, multiple displacement amplified DNA derived from 20 K. pneumoniae clinical isolates with a blaSHV-like genotype was used in a universal SHV PCR assay using SP6- (forward) and T7- (reverse) sequence-tagged primers. Identification and differentiation of blaSHV, blaLEN and blaOKP genes was obtained by bi-directional amplicon sequencing using SP6- and T7-specific primers. Three well characterised K. pneumoniae strains having a SHV-genotype were included in the study. The bi-directional amplicon sequencing, covering 800 bp (93%) of the blaSHV, blaLEN and blaOKP enzyme encoding sequences, allowed for an unequivocal discrimination of SHV, LEN and OKP genes. Moreover, sequencing revealed the presence of blaSHV allelic variants in six K. pneumoniae isolates in which the amplicons had to be cloned accordingly. Based on deduced amino-acid sequences, a dendrogram was constructed. Seventeen out of 20 K. pneumoniae isolates with an ESBL-phenotype formed a SHV-like cluster, two were LEN-like, and one isolate was OKP-like. The PCR-based molecular typing method described here enables a rapid, reliable and cost-effective identification and differentiation of blaSHV, blaOKP and blaLEN genes.Original Publication: Maria Tärnberg, Lennart E. Nilsson and Hans-Jürg Monstein, Molecular identification of blaSHV, blaLEN and blaOKP β-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons, 2009, Molecular and Cellular Probes, (23), 3-4, 195-200. http://dx.doi.org/10.1016/j.mcp.2009.01.005 Copyright: Elsevier Science B.V., Amsterdam http://www.elsevier.com/</p

Antimicrobial activity against a global collection of skin and skin structure pathogens: results from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.), 2010–2014

Background: As part of the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) we report antimicrobial resistance among Gram-positive and Gram-negative isolates collected globally from integumentary sources between 2010 and 2014. Methods: Minimum inhibitory concentrations and antimicrobial resistance were determined according to Clinical and Laboratory Standards Institute guidelines (US Food and Drug Administration breakpoints against tigecycline). The Cochran–Armitage trend test was used to identify statistically significant changes in resistance. Results: Global rates of methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii were 38% and 43%, respectively. No S. aureus isolates were resistant to linezolid or vancomycin; all isolates were susceptible to tigecycline. Two percent of Enterococcus faecalis and 28% of Enterococcus faecium were vancomycin-resistant. Extended-spectrum β-lactamase (ESBL) producers accounted for 22% of Klebsiella pneumoniae and 16% of Escherichia coli. Resistance to minocycline among E. faecalis, E. faecium, K. pneumoniae, and E. coli decreased significantly (p < 0.0001). There were significant increases (p < 0.0001) in A. baumannii resistance to cefepime, ceftazidime, ceftriaxone, levofloxacin, meropenem, and piperacillin–tazobactam. Conclusions: Among isolates from integumentary sources, rates of MRSA and ESBL-producing Enterobacteriaceae are stabilizing. Carbapenems and tigecycline have retained their in vitro activity against Gram-positive and Gram-negative organisms. Few agents were active against A. baumannii; its increasing resistance is cause for concern

Psysical and Mental Stress effect on the Employees of State Fire and Resource Service

Darba pirmajā daļā balstoties uz literatūras datiem un avotiem aprakstīti būtiskākie ugunsdzēsēju glābēju darba vides riska faktori un to iedarbība uz veselību. Ir veikta nelaimes gadījumu statistikas analīze izmantojot dinamiskās rindas jēdzienu, kā arī analizēta galveno notikumu statistika, kas ietekmē nelaimes gadījumus. Darba otrā daļā aprakstītas darba vides risku novērtēšanas metodes. Darba vides risku novērtēšanai izmantotas kvalitatīvas un puskvantitatīvas novērtēšanas metodes, kā arī veikta ugunsdzēsēju glābēju asinsspiedienu mērīšana dežūras laikā, dažādos posmos, noskaidrots darbinieku ķermeņa masas indekss un fiziskās darbspējas izmantojot Harvarda step-testu, izmantots sirds ritma monitors, noteikts veloergonometrijas tests. Darba trešajā daļā aprakstīti pētījuma rezultāti un dots to iztirzājums. Secināts, ka ugunsdzēsēju glābēju darbs ir vidēji smags – smags darbs, kur vislielākā slodze ir mugurai, rokām un pleciem, un ir liels stresa līmenis.The first part of the basis of literature data and sources are described in the main fire rescuers work environment risk factors and their effects on health. Is the incident statistical analysis of the series concept, as well as the analysis of the main event statistics affecting accidents. In the second part describes the risk assessment methods. To risk assessment used qualitative and semi-quantitative assessment methods, as well as in fire rescuers blood pressure measurement on-call during the different stages of the employees found the body mass index and physical ability to work with Harvard step-test is used to monitor heart rate, defined bicycle ergometry. The third part describes the results of the study and given the discussion. Concluded that the fire rescuers work is moderate - hard work, where the greatest load of the back, arms and shoulders, and has a high level of stress. Developed practical recommendations

Travel-associated faecal colonization with ESBL-producing Enterobacteriaceae : incidence and risk factors

Objectives To study the acquisition of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) among the faecal flora during travel, with a focus on risk factors, antibiotic susceptibility and ESBL-encoding genes. Methods An observational prospective multicentre cohort study of individuals attending vaccination clinics in south-east Sweden was performed, in which the submission of faecal samples and questionnaires before and after travelling outside Scandinavia was requested. Faecal samples were screened for ESBL-PE by culturing on ChromID ESBL and an in-house method. ESBL-PE was confirmed by phenotypic and genotypic methods. Susceptibility testing was performed with the Etest. Individuals who acquired ESBL-PE during travel (travel-associated carriers) were compared with non-carriers regarding risk factors, and unadjusted and adjusted ORs after manual stepwise elimination were calculated using logistic regression. Results Of 262 enrolled individuals, 2.4% were colonized before travel. Among 226 evaluable participants, ESBL-PE was detected in the post-travel samples from 68 (30%) travellers. The most important risk factor in the final model was the geographic area visited: Indian subcontinent (OR 24.8, P &lt; 0.001), Asia (OR 8.63, P &lt; 0.001) and Africa north of the equator (OR 4.94, P  = 0.002). Age and gastrointestinal symptoms also affected the risk significantly. Multiresistance was seen in 77 (66%) of the ESBL-PE isolates, predominantly a combination of reduced susceptibility to third-generation cephalosporins, trimethoprim/sulfamethoxazole and aminoglycosides. The most common species and ESBL-encoding gene were Escherichia coli (90%) and CTX-M (73%), respectively. Conclusion Acquisition of multiresistant ESBL-PE among the faecal flora during international travel is common. The geographical area visited has the highest impact on ESBL-PE acquisition.Funding Agencies|Medical Research Council of Southeast Sweden|FORSS-12368FORSS-36511FORSS-87551|ALF grants, Ostergotland County Council|LIO-10885LIO-16741LIO-61341LIO-127281|</p

Duration of travel-associated faecal colonisation with ESBL-producing Enterobacteriaceae - A one year follow-up study

In a previous study, we found that 30% of individuals travelling outside Scandinavia acquired extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) in their faecal flora. The aim of this study was to determine the duration of travel-associated faecal colonisation with ESBL-PE, to assess risk factors for prolonged colonisation and to detect changes in antibiotic susceptibility during prolonged colonisation

Varying High Levels of Faecal Carriage of Extended-Spectrum Beta-Lactamase Producing Enterobacteriaceae in Rural Villages in Shandong, China: Implications for Global Health

Antibiotic resistance is considered a major threat to global health and is affected by many factors, of which antibiotic use is probably one of the more important. Other factors include hygiene, crowding and travel. The rapid resistance spread in Gram-negative bacteria, in particular extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae (ESBL-E), is a global challenge, leading to increased mortality, morbidity and health systems costs worldwide. Knowledge about resistance in commensal flora is limited, including in China. Our aim was to establish the faecal carriage rates of ESBL-E and find its association with known and suspected risk factors in rural residents of all ages in three socio-economically different counties in the Shandong Province, China. Faecal samples and risk-factor information (questionnaire) were collected in 2012. ESBL-E carriage was screened using ChromID ESBL agar. Risk factors were analysed using standard statistical methods. Data from 1000 individuals from three counties and in total 18 villages showed a high and varying level of ESBL-E carriage. Overall, 42% were ESBL-E carriers. At county level the carriage rates were 49%, 45% and 31%, respectively, and when comparing individual villages (n = 18) the rate varied from 22% to 64%. The high level of ESBL-E carriage among rural residents in China is an indication of an exploding global challenge in the years to come as resistance spreads among bacteria and travels around the world with the movement of people and freight. A high carriage rate of ESBL-E increases the risk of infection with multi-resistant bacteria, and thus the need for usage of last resort antibiotics, such as carbapenems and colistin, in the treatment of common infections.Funding Agencies|National Nature Science Foundation of China [71073098]; Swedish International Development Cooperation Agency [2010-001861]; Swedish Research Council [2013-46520-109134-94]; China Medical Board [11-068]</p