Helena Sousa

Luisa Peixe

Sandra Quinteira

Open Repository of the University of Porto

Conclusion: The study led to identification of a number of
transcripts showing over-expression in drug resistant isolates,
some of which corresponded to known genes like PSA-2,
nucleoside transporter (NT), tetracycline resistant protein
homologue (TRH) and proteasome regulatory ATPases. Three
clones corresponded to the PSA-2, a glycoprotein with leucine
rich repeats that has a role in facilitating attachment and
internalization of parasites by macrophages, resisting
complement mediated cell lysis. NT and TRH may promote
resistance to drug by inhibiting the drug intake by the parasite.
Proteasome and associated ATPases help increased survival of
Mycobacterium inside macrophage by lending resistance to
nitric oxide. The over-expression of this protein in drug resistant
Leishmania may help the parasite to persist in host. The study
establishes the utility of genome-wide RNA expression profiling
in Leishmania and identifies numerous genes with potential role
in drug resistance.
Emergence and molecular evolution of antibiotic resistance
O205
A multidimensional screening of non-clinical
isolates reveals metallo-beta-lactamases in the
environment
S. Quinteira, H. Ferreira, L.V. Peixe (Porto, P)
Objectives: Even though increasingly reported in many
countries, metallo-beta-lactamase (MBL)-producing Gram-
negative bacilli have been associated to the hospital setting,
suggesting a relative confinement within this ecological niche. In
this study, the presence of MBLs and imipenem susceptibility in
Gram-negative bacilli were evaluated among non-clinical
sources in order to verify a possible spreading into new,
previously undescribed, environments.
Methods: Samples (24 from healthy human volunteer faeces,
30 from poultry skin, 4 from swine faeces, 13 from rivers and
29 from hospital wastewaters-receiving urban sewage) were
collected in Northern Portugal between 2001 and 2004. Sample
aliquots were platted, in MacConkey agar and/or Pyocyanosel
agar supplemented with imipenem (2 mg/L) and colonies with
different morphology were collected. Species identification was
performed with the API 32GN. Imipenem susceptibility was
determined using the disk diffusion method. A bioassay
method was employed in order to detect MBL production
and positive results were later confirmed by a multiplex PCR
assay designed to amplify both blaVIM and blaIMP genes.
Characterisation of MBL-type was possible through
sequencing.
Results: 190 Gram-negative bacilli (21 lactose-fermenters and
169 non lactose-fermenters) were isolated from different sources.
Decreased susceptibility to imipenem was observed in 64
isolates from sewage (n = 25), rivers (n = 17), swines (n = 12)
and healthy volunteers (n = 10). All poultry isolates showed
imipenem susceptibility. Positive bioassays were detected in 49
out of the 64 isolates with reduced susceptibility to imipenem.
blaVIM-2 gene was detected in three isolates: two Pseudomonas
aeruginosa (river and hospital sewage) and a P. alcaligenes
(hospital sewage). blaIMP was not detected in none of the
isolates.
Conclusions: Carbapenems are in many cases the last
therapeutic resort and the detection of carbapenem-
hydrolyzing MBL producers in the hospital setting is a
worrisome phenomenon. Although VIM and IMP-producing
strains were not observed in animals and human healthy
volunteers, these enzymes seem not to be restricted to the
hospital setting anymore. Disturbingly, the results emerging
from this study show that, even if in very low frequencies, the
presence of VIM-2 loose in the environment, namely in sewage
and rivers, may undermine the effectiveness of carbapenems,
largely depending on the eradication or confinement of resistant
bacteria.
O206
CTX-M-producing Escherichia coli now the
dominant cephalosporin-resistant
Enterobacteriaceae in London and SE England
N. Potz, R. Hope, M. Warner, A.P. Johnson, D. Livermore on
behalf of the London & South East ESBL Project Group
Objectives: There is increasing concern about the spread of
Enterobacteriaceae, principally E. coli, producing CTX-M-type
extended-spectrum beta-lactamases (ESBLs) in the UK. This is
reflected in increasing surveillance reports and submissions to
the reference laboratories, with many isolates reportedly from
community patients. To better assess the situation, a prospective
survey of cephalosporin-resistance was undertaken for
Enterobacteriaceae across London and Southeast England.
Methods: Eighteen laboratories participated: 10 in London and
8 elsewhere in southeast England. These screened all
Enterobacteriaceae isolates using cefpodoxime or both
cefotaxime and ceftazidime. Isolates resistant to any of these 3
cephalosporins were submitted for centralised susceptibility
testing and identification. Interpretive reading was performed,
allowing an inference of resistance mechanism(s).
Results: During a 12–week collection period, >1000
cephalosporin-resistant Enterobacteriaceae isolates were
collected, mostly submitted on the basis of their cefpodoxime
resistance. E. coliwas by far the most numerous species (>50% of
all isolates), with over 70% of these having an ESBL, usually
conferring a CTX-M phenotype. Klebsiella and Enterobacter spp.
were also well represented (each over 15% of resistant isolates).
The majority of the resistant Klebsiella isolates had phenotypes
indicative of ESBL production but two-thirds of enterobacters
had phenotypes implying derepression of AmpC. ESBLs were
detected in a few Citrobacter, Morganella and Serratia spp.
isolates, but not in P. mirabilis. Approximately 60% of all
cephalosporin-resistant Enterobacteriaceae were inferred to be
ESBL-producers, mostly with CTX-M phenotypes, whereas
isolates with AmpC phenotypes accounted for 20%. The
remaining 20% comprised of isolates inferred to have other
beta-lactamases, or non-enzymatic resistance.
Conclusion: Until 2000 CTX-M-beta-lactamases were unknown
in the UK and cephalosporin-resistant Enterobacteriaceae were
mostly enterobacters with AmpC or Klebsiella spp. with TEM or
SHV ESBLs. This survey shows a dramatic shift towards E. coli
as the main host for ESBLs and cephalosporin resistance, and
towards CTX-M phenotypes.
Abstracts
48


English

A multidimensional screening of non-clinical isolates reveals metallo-beta-lactamases in the environment

Repositório Aberto da Universidade do Porto

Conclusion: The study led to identification of a number oftranscripts showing over-expression in drug resistant isolates,some of which corresponded to known genes like PSA-2,nucleoside transporter (NT), tetracycline resistant proteinhomologue (TRH) and proteasome regulatory ATPases. Threeclones corresponded to the PSA-2, a glycoprotein with leucinerich repeats that has a role in facilitating attachment andinternalization of parasites by macrophages, resistingcomplement mediated cell lysis. NT and TRH may promoteresistance to drug by inhibiting the drug intake by the parasite.Proteasome and associated ATPases help increased survival ofMycobacterium inside macrophage by lending resistance tonitric oxide. The over-expression of this protein in drug resistantLeishmania may help the parasite to persist in host. The studyestablishes the utility of genome-wide RNA expression profilingin Leishmania and identifies numerous genes with potential rolein drug resistance.Emergence and molecular evolution of antibiotic resistanceO205A multidimensional screening of non-clinicalisolates reveals metallo-beta-lactamases in theenvironmentS. Quinteira, H. Ferreira, L.V. Peixe (Porto, P)Objectives: Even though increasingly reported in manycountries, metallo-beta-lactamase (MBL)-producing Gram-negative bacilli have been associated to the hospital setting,suggesting a relative confinement within this ecological niche. Inthis study, the presence of MBLs and imipenem susceptibility inGram-negative bacilli were evaluated among non-clinicalsources in order to verify a possible spreading into new,previously undescribed, environments.Methods: Samples (24 from healthy human volunteer faeces,30 from poultry skin, 4 from swine faeces, 13 from rivers and29 from hospital wastewaters-receiving urban sewage) werecollected in Northern Portugal between 2001 and 2004. Samplealiquots were platted, in MacConkey agar and/or Pyocyanoselagar supplemented with imipenem (2 mg/L) and colonies withdifferent morphology were collected. Species identification wasperformed with the API 32GN. Imipenem susceptibility wasdetermined using the disk diffusion method. A bioassaymethod was employed in order to detect MBL productionand positive results were later confirmed by a multiplex PCRassay designed to amplify both blaVIM and blaIMP genes.Characterisation of MBL-type was possible throughsequencing.Results: 190 Gram-negative bacilli (21 lactose-fermenters and169 non lactose-fermenters) were isolated from different sources.Decreased susceptibility to imipenem was observed in 64isolates from sewage (n = 25), rivers (n = 17), swines (n = 12)and healthy volunteers (n = 10). All poultry isolates showedimipenem susceptibility. Positive bioassays were detected in 49out of the 64 isolates with reduced susceptibility to imipenem.blaVIM-2 gene was detected in three isolates: two Pseudomonasaeruginosa (river and hospital sewage) and a P. alcaligenes(hospital sewage). blaIMP was not detected in none of theisolates.Conclusions: Carbapenems are in many cases the lasttherapeutic resort and the detection of carbapenem-hydrolyzing MBL producers in the hospital setting is aworrisome phenomenon. Although VIM and IMP-producingstrains were not observed in animals and human healthyvolunteers, these enzymes seem not to be restricted to thehospital setting anymore. Disturbingly, the results emergingfrom this study show that, even if in very low frequencies, thepresence of VIM-2 loose in the environment, namely in sewageand rivers, may undermine the effectiveness of carbapenems,largely depending on the eradication or confinement of resistantbacteria.O206CTX-M-producing Escherichia coli now thedominant cephalosporin-resistantEnterobacteriaceae in London and SE EnglandN. Potz, R. Hope, M. Warner, A.P. Johnson, D. Livermore onbehalf of the London & South East ESBL Project GroupObjectives: There is increasing concern about the spread ofEnterobacteriaceae, principally E. coli, producing CTX-M-typeextended-spectrum beta-lactamases (ESBLs) in the UK. This isreflected in increasing surveillance reports and submissions tothe reference laboratories, with many isolates reportedly fromcommunity patients. To better assess the situation, a prospectivesurvey of cephalosporin-resistance was undertaken forEnterobacteriaceae across London and Southeast England.Methods: Eighteen laboratories participated: 10 in London and8 elsewhere in southeast England. These screened allEnterobacteriaceae isolates using cefpodoxime or bothcefotaxime and ceftazidime. Isolates resistant to any of these 3cephalosporins were submitted for centralised susceptibilitytesting and identification. Interpretive reading was performed,allowing an inference of resistance mechanism(s).Results: During a 12–week collection period, >1000cephalosporin-resistant Enterobacteriaceae isolates werecollected, mostly submitted on the basis of their cefpodoximeresistance. E. coliwas by far the most numerous species (>50% ofall isolates), with over 70% of these having an ESBL, usuallyconferring a CTX-M phenotype. Klebsiella and Enterobacter spp.were also well represented (each over 15% of resistant isolates).The majority of the resistant Klebsiella isolates had phenotypesindicative of ESBL production but two-thirds of enterobactershad phenotypes implying derepression of AmpC. ESBLs weredetected in a few Citrobacter, Morganella and Serratia spp.isolates, but not in P. mirabilis. Approximately 60% of allcephalosporin-resistant Enterobacteriaceae were inferred to beESBL-producers, mostly with CTX-M phenotypes, whereasisolates with AmpC phenotypes accounted for 20%. Theremaining 20% comprised of isolates inferred to have otherbeta-lactamases, or non-enzymatic resistance.Conclusion: Until 2000 CTX-M-beta-lactamases were unknownin the UK and cephalosporin-resistant Enterobacteriaceae weremostly enterobacters with AmpC or Klebsiella spp. with TEM orSHV ESBLs. This survey shows a dramatic shift towards E. colias the main host for ESBLs and cephalosporin resistance, andtowards CTX-M phenotypes.Abstracts48

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A multidimensional screening of non-clinical isolates reveals metallo-beta-lactamases in the environment

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