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The bioMérieux solution
Did you know?
VITEK2 has been challenged with ESBL in
http://www.lahey.org/studies
Site of Lahey Clinic, where tables are updated for

several studies.The broader scope has been
B-lactamases with amino-acid sequences
published by Livermore et al.
120 TEM
50 OXA
12 CMY

Multicentre Evaluation of the VITEK 2 Advanced Expert System for interpretive reading of antimicrobial resistance tests.
6 VIM
Livermore et al. Journal of Antimicrobial Chemotherapy (2002) 49,289-30010 European centers results were compared final result to final The study was performed by 10 European centers from nine different The Hall Laboratory of Experimental Evolution
European countries, and involved around 1000 strains.
test agreement resistant Klebsiella spp 99 E. cloacae, C. freundii Identifying Resistance E. gergoviae total 137
bioMérieux UK jointly organised a symposium on Klebsiella spp 5 Identifying Resistance, last February in London,
with the Public Health Laboratory Service (PHLS).
The only way to evaluate the performance of an expert system is to One hundred and twenty people attended 8 lectures.
compare final results.
Here the comparison was made between results after interpretation by the expert system, and genotypic findings or human expert results The first part of the meeting addressed the new when using a phenotypic method (S.pneumoniae).
Health Organisation in this country (replacement ofthe PHLS by the Health Protection Agency (HPA)), As a consequence, computation of results was similar to that of bringing expertise and excellence to the National Health Service (NHS), through a new organisation • Agreement when both experts were giving the same result.
and a series of reference labs. The focus of the • Disagreement when they differed.
presentations was epidemiology and microbiology • Low discrimination when VITEK2 expert was proposing 2 or 3 answers, one of them being right.
and key speakers addressed the audience.
The second part of the meeting concerned the control of antibiotics in hospitals, the role of themicrobiology laboratory in detecting resistance andhow this can aid infection control by more rapidreporting using VITEK®2.
In the final session, Dr David Livermore discussed "Green cats" and the need for interpretive reading of antibiotic results. Dr Jean-Pierre Marcel frombioMérieux, concluded the meeting by discussing the company's experience in developing expert systems and the current developments in DNA Chip technology.
In the UK, authorities are working on Infection Anne Beal,
Control and Resistance Detection based on the Microbiology Laboratory Manager,
House of Lords white paper (Path to Least (Fort Lauderdale, Florida) resistance, 1998) and the Department of Health surrounded by 4 VITEK® 2 XL report "Getting ahead of the Curve", in order to instruments at IntegratedRegional Laboratories (IRL).
reduce the Socio-Economic Burden of Hospital This lab serves 13 hospitals in South Florida and processes 2000 samples every day.
bioMérieux UK is closely supporting these official VITEK 2 was chosen for routine use at this core lab facility bodies, as was the case with this symposium.
because of its automation, rapid results and the AdvancedExpert System.
Did you know?
What is the impact of ESBL?
ESBL-producing bacteria escape treatment by
nccls recommendations Cephalosporins, including widely used ref: M100-S13 (M7) January 2003 Cephalosporins III and IV.
Table 2A Enterobacteriaceae
Comment 6
Why look for ESBL?
Strains of Klebsiella spp and E.coli that produce extended spectrum beta-lactamase (ESBLs) may be Expression of ESBL production is variable in clinically resistant to therapy with penicillins, cephalosporins, or aztreonam, despite apparent in vitro intensity and with substrates. That means that susceptibility to some of these agents.
Some of these strains will show MICs above the normal susceptible population but below the standard the test result with a drug can be corrected breakpoints for certain extended-spectrum cephalosporins or aztreonam. Such strains should be when ESBL production has been demonstrated.
screened for potential ESBL production by using the ESBL screening breakpoints before reportingresults for penicillins, extended-spectrum cephalosporins, or aztreonam.
Other strains may test intermediate or resistant by standard breakpoints to one or more of these agents.
How to suspect an ESBL?
In all strains with ESBLs, the MICs for one or more of the extended-spectrum cephalosporins or By testing several cephalosporins to contourn aztreonam should decrease in the presence of clavulanic acid as determined in phenotypic substrate specificity. Any non-susceptible result confirmatory testing.
For all confirmed ESBL-producing strains, the test interpretation should be reported as resistant to all for cefotaxime, ceftazidime, aztreonam or penicillins, cephalosporins, and aztreonam.
cefpodoxime is a strong indication with some The decision to perform ESBL screening tests on all urine isolated should be made on an institutional basis, considering prevalence, therapy, and infection-control issues.
What is the reference method?
Screening and Confirmatory Tests for ESBLs in
Amplification then sequencing of resistant genes Klebsiella pneumoniae, K.oxytoca, and Escherichia coli.
is the reference to identify mutations turning Medium (CAMHB), antibiotic concentrations, standard broth dilution recommendations for inoculum, incubation conditions, incubation length.
some ß-lactamases into ESBL.
Growth may indicate ESBL production.
Phenotypic confirmatory tests are more widely Recommended drugs are: used: restoration of ß-lactam activity by cefpodoxime (4 µg/mL), ceftazidime, aztreonam, cefotaxime, ceftriaxone (1 µg/mL).
ß-lactamase inhibitors such as clavulanic acid.
These can be performed using the diffusion method (double disk method) or dilution Sub-classes of cephem (parenteral) class cefazolin, cephalothin, cephapirin, cephradin cephalosporins II cefamandole, cefonicid, cefuroxim (sodium) cephalosporins III cefoperazone, cefotaxime, ceftazidime, ceftizoxime, ceftriaxone What antibiotics to use for
cephalosporins IV ne of its subsidiaries / Printed in F cefmetazole, cefotetan, cefoxitin Alternative drugs are mainly carbapenems (imipenems, meropenem.) or combinations of Cephalosporins I, II, III, IV are sometimes referred to as 1st, 2nd, 3rd, and 4th generation ß-lactams with inhibitors of ß-lactamase or Cephalosporins III and IV are also referred to as "extended-spectrum cephalosporins". This does not imply activity against ESBL-producing gram-negative bacteria.
For all confirmed ESBL-producing strains, the test interpretation should be reported as resistant for this antimicrobial class or subclass.
ves the right to modify specifications without noti Extended spectrum ß-lactamase ESBL + impermeability (cephamycins) INTERNATIONAL NEWSLETTER ITEK are registered and protected trademarks belonging to bioMérieux s Director of publications : Thierry Bernard for more information : identifying.resistance@eu.biomerieux.com bioMérieux sa69280 Marcy l'EtoileFrance 9003E / This document is not legally binding. bioMérieux reser Tel. (33) 04 78 87 20 00 Fax (33) 04 78 87 20 90 bioMérieux, blue logo, Identifying Resistance and V



n° 4 • December 2003 Did you know?
IDENTIFYING RESISTANCE Newsletter, bioMérieux's ambition is to contribute to ■ What is the impact the awareness and progress in the field of resistance to antibiotics.
■ Why look for ESBL? I hope the information, papers written ■ How to suspect by worldwide specialists, brings you valuable data to help you in your reference method? activities and day-to-day practice.
■ What antibiotic This new issue deals with a complex resistance mechanism that appeared less than twenty years ago and for which bioMérieux rapidly adapted its offer of tests and software.
This is a perfect illustration of our commitment and continuous effort to bring you a global offer in terms of instruments, reagents, software and in Enterobacteriaceae expert systems.
We will do our best to deserve your confidence and continue to propose Karen Bush, Ph.D., is the Team Leader for the Biology Antimicrobial innovative new products to help you in Agents Research Team at Johnson & Johnson Pharmaceutical Research & your endeavour.
Development (Raritan, NJ, USA), where she is responsible formicrobiology research in Drug Discovery. Her work on beta-lactamase Dr. Benoît Adelus inhibitors and resistance mechanisms contributed to proposing an Chief Executive Officer updated functional classification scheme for these enzymes. Karen Bush Development of the "third generation" its single amino acid variant TEM-2, and cephalosporins in the early 1980s was the functionally similar SHV-1 enzyme, based heavily on the ability of these agents together with the oxacillin-hydrolyzing to escape hydrolysis by all the common OXA-1 enzyme, were the most common ß-lactamases in both Gram-positive and plasmid-encoded ß-lactamases in Gram- Gram-negative bacteria (18). Broad n e g a t i v e b a c t e r i a a c c o r d i n g t o
spectrum ß-lactamases with the ability to
epidemiological surveys in the 1980s hydrolyze the most common penicillins (13). However, the new cephalosporins,
and cephalosporins had been identified in cefotaxime, ceftazidime and ceftriaxone, virtually all species of Enterobacteriaceae and the monobactam aztreonam exhibited before 1980 (19), and had begun to
good antibacterial activity against Gram- appear in large numbers of Haemophilus negative bacilli, in part because of their influenzae and Neisseria gonorrhoeae exceptional stability to the infamous TEM, isolates (2). The broad spectrum TEM-1,
SHV and OXA enzymes (6).
Identifying Resistance International Newsletter • December 2003 the seeds of better health ESBL in Enterobacteriaceae
To the dismay of their developers, these Coincidentally, the first ESBL-producing agents were challenged by an unexpected K. pneumoniae isolates from the United set of mutational events shortly after their States were all identified during the first six introduction into clinical medicine. The first months of 1987 in Boston, New York City, extended spectrum ß-lactamases (ESBLs) Chicago and California, but with a were reported from Germany in 1983 with ceftazidime-resistant phenotype (8, 14,
the description of three independent 15, 20). In all cases, the producing
K. pneumoniae isolates from the same organisms were multidrug resistant due to hospital exhibiting transferable cefotaxime large plasmids that usually included resistance (10). Retrospectively, an even
aminoglycoside resistance determinants as earlier Argentinian K. pneumoniae isolatewas later shown to produce an ESBL in well as ß-lactamase genes.
1982, the year after the introduction of Phenotypically, a double disk diffusion cefotaxime in the Americas (12).
Major outbreaks of ESBL-producing amoxicillin-clavulanic acid was used to Enterobacteriaceae were first reportedfrom France, where 283 cefotaxime- identify the presence of early ESBLs in resistant K. pneumoniae isolates were E. coli and K. pneumoniae (9). The fact
detected from 1984 through June 1987, in that ESBLs respond to inhibition by either addition to another 200 isolates of E. coli, clavulanic acid or tazobactam has served Enterobacter spp., Serratia marcescens, as a distinguishing characteristic of these K. oxytoca and Citrobacter freundii that enzymes throughout their history (6) and
produced the same ESBL (16).
is the basis of the NCCLS protocol for
detection of ESBLs in E. coli and
K. pneumoniae (17).
Characteristics of ESBLs
Data compiled from http://www.lahey.org/studies/webt.stm. (February, 2003).
Enzyme family
Total number in family 73 TEM-1 variants 32 SHV-1 variants 19 TEM-2 variants* 13 SHV-2 variants* 7 OXA-10 variants Amino acids in enzyme, including leadersequenceNumber of amino acid positions at which may differ 20-25% substitutions have beenreported from enzymesin clinical isolatesMaximum number of mutations in a singleESBL comparedto parentMost common OXA-10 series: I10T, G20S, T110S, substitutions in mature R164S or R164H(N=25) G238S or G238A (N = 17) Y184F, E240G, S258S, *TEM-1 and TEM-2, differing by a Q39K substitution are not considered to be ESBLs. SHV-1 differs from SHV-2 by a G238S substitution, rendering SHV-2 an ESBL.
#Each substitution appears in 3 enzymes. Different combinations are observed.


States, almost all the early ESBLs were
identified as TEM variants, whereas in
Europe a mixture of TEM and SHV ESBLs
were characterized (12).
Comparisons of the geographically diverseESBLs indicated that two distinct ESBLpopulations were evident. In Europe, oneset of enzymes showed preferentialhydrolysis of cefotaxime compared toceftazidime and were initially named "CTX"enzymes; a second set of enzymespreferentially hydrolyzed ceftazidime andwere named "CAZ" enzymes. However,upon sequencing of the producing genes,it was noted that both sets of enzymeswere derived from the blaTEM-1 gene.
Thus, an early consensus was reached in
the ß-lactamase community that the
ESBLs would be numbered according to
their parent, and not according to their
functional status (5). ESBL nomenclature
is currently being monitored on a website
managed by G. A. Jacoby and K. Bush
(http://www.lahey.org/studies/webt.stm),
where amino acid sequences and
literature references are provided for
all TEM and SHV variants, and for
OXA-derived ESBLs (see Table). In
addition, references are given for all OXA,
CMY-type, IMI-type and CTX-M sequences.
Resistance by ESBL:
enzymatic inactivation
Of assistance to the practicing laboratory enzymologist is a table of all isoelectricpoints reported for all ESBLs.
Although the majority of ESBLs areassociated with either a TEM or SHVheritage, other enzyme families haveachieved recognition as they become As improved molecular techniques predominant in their own geographical became more widely available in the niches. Extended spectrum OXA-derived 1990s, sequencing of the responsible enzymes were originally reported in genes became routine and is considered Pseudomonas aeruginosa isolates from to be the "gold standard" for ESBL Turkey (7) and have now been identified
identification (2). The original ESBLs from
from other European sites (2).
Europe and the United States were allderived from the common TEM-1 andSHV-1 enzymes, usually differing by one to One of the most rapidly growing new three amino acid substitutions compared families of ESBLs is the CTX-M family, to the parent enzyme. In almost all cases, CTX-M-1 was first identified in cefotaxime- these changes were due to point mutations resistant K. pneumoniae isolates from in the nucleotide sequences. In the United Western Europe; CTX-M-2 was then found


Bauernfeind, A., I. Stemplinger, R. Jungwirth, S. Ernst, and J. M.
Casellas. 1996. Sequences of ß-lactamase genes encoding CTX-M-1(MEN-1) and CTX-M-2 and relationship of their amino acidsequences with those of other ß-lactamases. Antimicrob. AgentsChemother. 40:509-513.
Bradford, P. A. 2001. Extended-spectrum ß-lactamases in the 21stcentury: characterization, epidemiology, and detection of thisimportant resistance threat. Clin. Microbiol. Rev. 14:933-951.
in several South American isolates and In addition, it is important to note that differed by 16% in its amino acid sequence Brenwald, N. P., G. Jevons, J. M. Andrews, J. H. Xiong, P. M.
Hawkey, and R. Wise. 2002. An outbreak of a CTX-M-type from CTX-M-1 (1). These enzymes
Enterobacteriaceae, with their production ß-lactamase-producing Klebsiella pneumoniae: the importance of strongly prefer cefotaxime as a substrate often masked by the concurrent using cefpodoxime to detect extended-spectrum ß-lactamases.J.
Antimicrob. Chemother. 51:195-196.
and hydrolyze ceftazidime poorly.
production of AmpC cephalosporinases Bush, K. 2001. ß-lactamases in gram-negative bacteria: diversity (4). With the promiscuous transfer of
and impact on the selection of antimicrobial therapy. Clin. Infect.
At this time there are over 25 unique ESBL determinants among Gram-negative Dis. 32:1085-1089.
members of this family. It is regarded as rods, we can only expect these enzymes Bush, K., and G. Jacoby. 1997. Nomenclature of TEM to continue to proliferate in the present ß-lactamases. J. Antimicrob. Chemother. 39:1-3.
the most prominent ESBL in SouthAmerica, and has now been identified with Bush, K., G. A. Jacoby, and A. A. Medeiros. 1995. A functionalclassification scheme for ß-lactamases and its correlation with outbreaks in China and the United molecular structure. Antimicrob. Agents Chemother. 39:1211- Kingdom (3). The producing organisms
do not appear to be resistant to Hall, L. M. C., D. M. Livermore, D. Gur, M. Akova, and H. E.
Akalin. 1993. OXA-11, an extended spectrum variant of OXA-10 ceftazidime in standard susceptibility (PSE-2) ß-lactamase from Pseudomonas aeruginosa. Antimicrob.
testing, so detection systems utilizing only Agents Chemother. 37:1637-1644.
ceftazidime will not identify a CTX-M ESBL Jacoby, G. A., A. A. Medeiros, T. F. O'Brien, M. E. Pinto, and H.
(3). As additional families of enzymes
Jiang. 1988. Broad-spectrum, transmissible ß-lactamases [letter].
N. Engl. J. Med. 319:723-723.
continue to be identified, it may be Jarlier, V., M. Nicolas, G. Fournier, and A. Philippon. 1988.
expected that even more narrow spectrum Extended broad-spectrum ß-lactamases conferring transferable ESBLs will become prevalent. resistance to newer ß-lactam agents in Enterobacteriaceae:Hospital prevalence and susceptibility patterns. Rev. Infect. Dis.
10:867-878.
10. Knothe, H., P. Shah, V. Krcmery, M. Antal, and S. Mitsuhashi.
1983. Transferable resistance to cefotaxime, cefoxitin,cefamandole and cefuroxime in clinical isolates of Klebsiella K. pneumoniae is often attributed solely pneumoniae and Serratia marcescens. Infection 11:315-317.
to ESBL production; however, other Livermore, D. 1995. ß-lactamases in laboratory and clinical factors must also be considered.
resistance. Clin. Microbiol. Rev. 8:557-584.
12. Medeiros, A. A. 1997. Evolution and dissemination of
ß-lactamases accelerated by generations of ß -lactam antibiotics.
The combined contributions of porin Clinic. Infect. Dis. 24(Suppl. 1):S19-45.
mutations, quantity of enzyme activity, and 13. Medeiros, A. A. 1989. Plasmid-determined ß-lactamases.
number of ß-lactamases per strain (4, 11)
Handbook of Experimental Pharmacology (91):101-127.
will result in elevated MICs for these 14. Naumovski, L., J. P. Quinn, D. Miyashiro, M. Patel, K. Bush, S. B.
Singer, D. Graves, T. Palzkill, and A. M. Arvin. 1992. Outbreak ofceftazidime resistance due to a novel extended-spectrumß-lactamase in isolates from cancer patients. Antimicrob.
Agents Chemother. 36(9):1991-1996.
15. Quinn, J. P., D. Miyashiro, D. Sahm, R. Flamm, and K. Bush.
1989. Novel plasmid-mediated ß-lactamase (TEM-10) conferring selective resistance to ceftazidime and aztreonam in clinical Enzymatic
isolates of Klebsiella pneumoniae. Antimicrob. Agents 16. Sirot, J., C. Chanal, A. Petit, D. Sirot, R. Labia, and G. Gerbaud.
1988. Klebsiella pneumoniae and other Enterobacteriaceae producing novel plasmid-mediated ß -lactamases markedly activeagainst third-generation cephalosporins: Epidemiologicalstudies. Rev. Infect. Dis. 10:850-859.
17. National Committee for Clinical Laboratory Standards. 2003.
Performance standards for antimicrobial susceptibility testing.
NCCLS approved standard M100-S13 (M7). National Committeefor Clinical Laboratory Standards, Wayne, PA.
18. Sykes, R. B., and K. Bush. 1983. Interaction of new cephalosporins
with ß-lactamases and ß-lactamase-producing Gram-negativebacilli. Rev. Infect. Dis. 5 Suppl. 2:S356-S366.
19. Sykes, R. B., and M. Matthew. 1976. The ß-lactamases of gram-
negative bacteria and their role in resistance to ß -lactam antibiotics.
J. Antimicrob. Chemother. 2:115-157.
20. Urban, C.,K. S. Meyer, N. Mariano, J. J. Rahal, R. Flamm, B. A.
Rasmussen and K. Bush. 1994. Identification of TEM-26ß-lactamase responsible for a major outbreak of ceftazidimeresistant Klebsiella pneumoniae. Antimicrob.
Agents Chemother. 38:392-395.

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37412_SpanishCover:37412_SpanishCover 9/17/09 10:37 AM Page 1 Publicado por la American Society for Reproductive Medicine, bajo la dirección del Comité de Educación del Paciente y el Comité de Publicaciones. Ninguna parte en este documento puede ser reproducida en ninguna forma sin permiso por escrito. Este folleto no pretende de ninguna manera sustituir, dictar ni definir

Mics4 preliminary findings

Monitoring the situation of children and women Multiple Indicator Cluster Survey SUMMARY REPORT Federal Republic of Nigeria National Bureau of Statistics MICS Nigeria, 2011 Summary Report In Memory This report is dedicated to the 24 people killed, and those who were injured, by the bomb attack on UN House in Abuja on the 26th August 2011. Amongst the people who lost their lives was Johnson Awotunde who devoted his time, energy and personal resources to the success of the survey until his untimely departure.