Importance of High Levels Detention by Enterococcus Resistance HLR to Aminoglycosides in the Treatme

1
Importance of High Levels Detention by
Enterococcus Resistance (HLR) to Aminoglycosides
in the Treatment of Serious Infections from
Hospital

• Marcelo M. Antunes1,2,3 Adriana A. Antunes4,5
• Galba M. Campos Takaki5
• 1Laboratório Central de Saúde Pública - LACEN,
  Recife/Brazil 2Hospital Barão de Lucena,
  Recife/Brazil 3Universidade Federal Rural de
  Pernambuco - UFRPE, Recife/Brazil 4Pós-Graduação
  em Ciências Biológicas UFPE, Recife/Brazil
  5Núcleo de Pesquisa em ciências Ambientais
  (NPCIAMB) - UNICAP, Recife/PE, Brazil Rua
  Fernandes Vieira, S/N, Boa Vista,
  Recife/PE/Brazil.
• Corresponding author mantunes_at_elogica.com.br
  adri_antunes_at_yahoo.com.br

2
Introduction

• Enterococcus spp. were first recognized as
  important ethiological agents of
  hospital-acquired infections in the middle of
  1970s. Their rising pathogenicity was probably
  due to the use of third-generation
  cephalosporins, to wich these bacteria are
  naturally resistant.
• They are Gram-positive, facultatively anaerobic
  cocci found in the gastrointestinal tract of most
  healthy humans and animals where they make up a
  significant portion of the normal microbiota.
  They are released into the environment via the
  sewage where they can survive for long periods of
  time.
• Enterococcus, in our days, represent the second
  leading cause of nosocomial urinary tract
  infections and the third leading cause of
  nosocomial bacteremia.

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• They include around 20 species, but most human
  enterococcal infections are caused by E. faecalis
  (80-90) and E. faecium (10-15). A few cases of
  human infections caused by other enterococci spp.
  such as E. durans, E. gallinarum and E.
  casseliflavus have also been reported.
• There are several types and many different
  mechanisms for Enterococcus resistance. The most
  relevant are the inherent resistance, including
  the resistance to stable penicillins and low
  levels of aminoglycoside resistance, in addition
  to the acquired resistance, which includes the
  HLR High Level of Resistance to
  aminoglycosides, characterized as a
  plasmid-mediated resistance caused by the
  expression of genes coding for enzymes that
  promote modifications in those antibiotics.
• The low level of the inherent or natural
  resistance to aminoglycosides by Enterococcus,
  difficults isolated treatments, although it
  allows their association to beta-lactam or to
  vancomicin, since those act at the cell wall
  level, promoting the aminoglycoside penetration.

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• This activity, however, is not observed when
  Enterococcus shows a high level of aminoglycoside
  resistance (HLR) through the acquisition of
  enzymes responsible for the resistance, stopping
  presenting the classic synergic action between
  the antimicrobial and the aminoglycoside, leading
  to the probable therapeutic failure during the
  treatment of infections caused by Enterococcus.
  Isolates with high-level resistance to STRP (MIC
  gt 2.000ug/ml) were first described in 1970, and,
  in 1979, strains highly resistant to GNT were
  reported.
• Such strains are not killed by penicillin plus
  the respective aminoglycoside therefore, to
  determine optimal therapy, clinically significant
  enterococci should be tested for high-level
  resistance to STRP and GNT. The abilities of
  commercial systems to detect high-level
  aminoglycoside resistance can vary.
• The synergy sensitivity screen (one well with
  2.000ug of GNT per ml and one with 2.000ug of
  STRP per ml) is the conventional formulation.
  This activity is not observed when the
  Enterococcus presents one high level of
  resistance to the aminoglycoside (HLR - High
  Level Resistance) with MIC above of 2.000ug/ml
  through the acquisition of responsible enzymes
  for the resistance.

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Objective

• This works primary objective is to demonstrate
  the importance of the detection of the high level
  of Enterococcus resistance (HLR) to the
  aminoglycoside with the purpose to evaluate the
  reliability of MicroScan rapid panels for
  detection of high-level resistance to
  aminoglycoside by enterococci. The synergies
  screen results provided by rapid and reformulated
  conventional MicroScan panels can offer a
  contribution to the treatment of severe
  infections caused by this microorganism.

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Materials and methods

• Microorganism
• A total of 40 Enterococcus faecalis samples
  recovered from patients receiving medical care in
  a public hospital located in the city of Recife,
  Pernambuco state, Brazil, were collected from
  March 2000 to September 2003. The reference
  strains used as control were Enterococcus
  faecalis ATCC 29212 (susceptible to vancomycin),
  Enterococcus faecalis SS 1332 (high level
  resistance to gentamicin and streptomycin, and
  vancomycin resistant) and Enterococcus faecium SS
  1274.

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Isolation and identification

• For the isolation of Enterococcus faecalis a
  conventional method was applied by seeding blood
  culture flasks to enriched media (Blood Agar
  Base) and selective-indicators (Enterococosel
  Agar). Enterococci were identified using standard
  methods based on gram staining characteristics.
  For the physiologic characterization the
  following biochemical tests were used catalase
  reaction, bile esculin, growth in 6.5 NaCl, LAP
  and PYR, besides automated tests with panels pos
  combo for identification, including 20
  biochemical tests.

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Microscan panels

• For HLR detection the sensitivity test was
  carried out by microdilution, using automated
  technology from MicroScan using gentamicin and
  streptomycin as standards, with MicroScan panels
  pos combo. Reformulated conventional (Pos Combo
  Type 6) and rapid (Pos Combo Type 1) panels were
  inoculated, incubated, and read according to the
  manufacturer's directions, except for the total
  incubation time for conventional panels that was
  48 h. Both panel types were incubated in and
  interpreted with the MicroScan WalkAway-96 system
  (Dade Behring) (Figure 1).
• Rapid panels were read at 3.5, 4.5, 5.5, 7, 8,
  11, and 15h the results were reported when
  growth in the control wells was satisfactory.
  Conventional panels were read with the WalkAway
  system after 18 h and then examined visually.

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• If one or both synergy screen wells showed no
  growth, the panel was re-incubated off-line at
  35C and read visually after incubation for a
  total period of 48 h.
• If a discrepancy existed between synergy screen
  results provided by the two panels or between
  results from a panel and the agar dilution, tests
  with both panels were repeated.
• The minimal inhibitory concentration (MIC) was
  determined by microdilution method with Microscan
  panels pos combo, CLSI cutoff points were used to
  interpret MIC data. CLSI considered strains with
  a MIC ? 2?g/ml as susceptible, those with a MIC
  4?g/ml as intermediate, and those with a MIC ?
  8?g/ml as resistant. Appropriate quality control
  was performed using Enterococcus faecalis and an
  investigational drug.

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Results and discussion

• From a total of 40 Enterococcus faecalis samples
  tested, 16 were sensible to streptomycin,
  gentamicin and no detectable levels of HLR for
  aminoglycoside were observed in 40 of the
  samples, 10 were resistant to streptomycin,
  gentamicin and HLR detected levels were observed
  for all aminoglycosides in 25 of the samples 8
  samples were resistant to streptomycin and
  sensitive to gentamicin, with HLR detectable
  levels only to streptomycin in 20 of samples,
  and 6 samples were sensitive to streptomycin and
  resistant to gentamicin, with detectable HLR
  levels to gentamicin and, consequently also to
  tobramycin and netilmicin in 15 of the samples
  studied, as shown in Table 1.
• According to our results, it is possible to
  conclude that the detection of Enterococcus
  strains showing high levels of resistance (HLR)
  is extremely important and must be included in
  the laboratorial routine, considering that these
  strains stop showing the classic synergic action
  between beta-lactams and aminoglycoside promoting
  failure of the treatment for severe infections,
  mainly hospital-acquired, caused by this
  microorganism.

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• Table 1 Results from HLR (High Level
  Resistance) detection by Enterococcus faecalis
  using automated methodology from MicroScan
  WalkAway-96 system (Dade Behring).

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Figure 1 Following steps of Enterococcus
isolation, identification and interpretation with
MicroScan WalkAway-96 automated system (Dade
Behring).
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Conclusions

• Considering the results obtained in the present
  study, it is possible to conclude that the
  detection of Enterococcus samples with high level
  of resistance (HLR) is extremely important and
  should be included in the laboratorial routine,
  specially taking into account that 60 of the
  samples studied, showed, at least, one type of
  HLR, indicating that those samples stopped
  providing the classic synergistic action between
  ?-lactams and aminoglycosides, promoting the
  failure in the treatment of severe infections,
  originated mainly from hospitals, caused by the
  afore mentioned microorganism.
• The presence of HLR for the two aminoglycosides
  markers was observed in 25 of the samples
  studied, what substantiate the concern in using
  the synergism of these these drugs.

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References

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