الفهرس 
Beta-LactamasesOnly 14 pages are availabe for public view
 |  | from | 191 |  |  |
| | | from | 191 | | |
Abstract
AmpC β-lactamases have gained importance since the
late ١٩٧٠s as one of the mediators of antimicrobial resistance in
Gram negative bacilli. These enzymes are typically produced by
isolates of E. coli, Klebsiella spp., Proteus, and Salmonella spp.
and are associated with multiple antibiotic resistance that leaves
few therapeutic options.
Enterobacteriaceae producing AmpC β-lactamases
(AmpC) have become a major therapeutic challenge. The
detection of AmpC-producing Klebsiella spp. is of significant
clinical relevance since AmpC producers may appear
susceptible to expanded-spectrum cephalosporins when initially
tested. This may lead to inappropriate antimicrobial regimens
and therapeutic failure. Thus, a simple and reliable detection for
AmpC producers is needed.
Cefoxtin screening test is reported as useful in screening
for AmpC. Modified Three Dimensional Test (M٣D) is
reported as simple and sensitive phenotypic detection method.
Many studies have validated the use of cloxacillin (as AmpC
inhibitor) to detect AmpC β-lactamases among Gram-negative
bacteria. Also, many studies have validated the use of
imipenem and cefoxitin as inducing agents to detect the
presence of inducible AmpC enzymes among enterobacterial
strains.Even though multiplex-PCR is a gold standard method to
detect AmpC β-lactamase genes (differentiating between
chromosomal and plasmid-mediated AmpC β-lactamases and
the different types or families of PMABLs), but the test is still
costly and time consuming and equipment availability is limited
to few laboratories, hence looking for specific and sensitive
phenotypic tests have always been a challenge.
Many clinical laboratories show interests in performing
phenotypic tests as these are cost effective, simple to perform,
easy to implement in the diagnostic laboratory to avoid
inappropriate therapy and for better infection control,
distinguishing between the cefoxitin-resistant AmpC producers
and cefoxitin-resistant non AmpC producers which may be due
to reduced membrane permeability and also, differentiation of
organisms expressing ESBLs from organisms expressing
plasmid-mediated AmpC β-lactamases in order to address
surveillance and epidemiology as well as hospital infection
control issues associated with these resistance mechanisms.
The aim of this work is to detect of the occurrence of
PMABLs in Klebsiella spp. isolates by phenotypic tests and
detect its gene types by multiplex-PCR.
This study was conducted on a total number of one
hundred clinical isolates of Klebsiella spp. collected from
different clinical specimens referred to the Central
Microbiological Laboratory of Ain Shams University Hospitals from September ٢٠١٢ to February ٢٠١٣. Isolates were screened
by Cefoxtin Disk resistance using Disk Diffusion Method
(DD). All isolates were subjected to seven phenotypic methods:
Cloxacillin Combined Disk Diffusion Test (CC-DD),
Cefoxitin-Cloxacillin Double-Disk Synergy Test (CC-DDS),
Ceftazidime-Imipenem Antagonism Test (CIAT), Cefoxitin-
Cefotaxime (FOX-CTX) Antagonism Test and Modified
Three Dimensional Test (M٣D) for detection of AmpC β-
lactamases. Detection of ESBLs production was done by using
Double Disk Synergy Test (DDS) and detection of Plasmidmediated
AmpC genes (ACC, FOX, MOX, DHA, CIT and
EBC) by Multiplex-PCR.
AmpC production was detected in ٥٧ (٥٧٪) out of the
١٠٠ isolates by M٣D test, ٤٤ (٧٧٫٢٪,) out of ٥٧ M٣D-positive
isolates were cefoxtin resistant with a sensitivity of ٧٧٫٢٪, a
specificity of ٩٥٫٣٪ and an excellent agreement between the
results of both tests (kappa=٠٫٧١٩). Thirteen (٢٢٫٨٪,) out of ٥٧
M٣D-positive isolates were positive by CC-DD test with more
specificity (٩٥٫٣٪) than sensitivity (٢٢٫٨٪) and a poor
agreement between the results of both tests (kappa=٠٫١٦٢)
while seven (١٢٫٣٪) out of ٥٧ M٣D-positive isolates were
positive by CC-DDS test with lower sensitivity (١٢٫٣٪), a
specificity of ٤١٫٩٪ and fair negative agreement between the
results of both tests (kappa= - ٠٫٤٢٨). Fourty-three (٧٥٫٤٪) out
of ٥٧ M٣D-positive isolates were only AmpC producers and ١٤ (٢٤٫٦٪) out of ٥٧ M٣D-positive isolates were ESBL/AmpC coproducers.
Plasmid-mediated AmpC β-lactamases were detected in
١٣ (٣٢٫٥٪) out of ٤٠ isolates by PCR. Only eight (٦١٫٥٪) out of
١٣ PCR-positive isolates were cefoxitin resistant with a
sensitivity of ٦١٫٥٪, a specificity of ٦٦٫٧٪ and week agreement
between the results of both tests (kappa=٠٫٢٦١). Eight (٦١٫٥٪)
out of ١٣ PCR-positive isolates were positive by CC-DD test
with more specificity (٨١٫٥٪) than sensitivity (٦١٫٥٪) and fair
agreement between the results of both tests (kappa=٠٫٤٣٠),
while five (٣٨٫٥٪) out of ١٣ PCR-positive isolates were
positive by CC-DDS test with less sensitivity (٣٨٫٥٪) than
specificity of ٧٧٫٨٪ and poor agreement between the results of
both tests (kappa=٠٫١٦٩). One (٧٫٧٪) out of ١٣ PCR-positive
isolates were positive by CIAT with lower sensitivity (٧٫٧٪),
higher specificity (٩٦٫٣٪) and poor agreement between results
of both tests (kappa=٠٫٠٥١). Two (١٥٫٤٪) out of ١٣ PCRpositive
isolates were positive by FOX-CTX antagonism test
with lower sensitivity (١٥٫٤٪), higher specificity (٨٨٫٩٪) and
poor agreement between the results of both tests
(kappa=٠٫٠٥١). Eight (٦١٫٥٪) out of ١٣ PCR-positive isolates
were only AmpC producers and five (٣٨٫٥٪) out of ١٣ PCRpositive
isolates were ESBL/AmpC co-producers.
Plasmid-mediated AmpC genes were detected in ١٣
(٣٢٫٥٪) out of ٤٠ isolates by Multiplex-PCR using familyspecific
primers. Eight (٦١٫٥٪) out of ١٣ PCR-positive isolates have the blaMOX gene, nine (٦٩٫٢٪) out of ١٣ PCR-positive
isolates have the blaCIT gene, five (٣٨٫٥٪) out of ١٣ PCRpositive
isolates have the blaFOX gene, five (٣٨٫٥٪) out of ١٣
PCR-positive isolates have the blaACC gene, one (٧٫٧٪) out of
١٣ PCR-positive isolates has the blaEBC gene and one (٧٫٧٪)
out of ١٣ PCR-positive isolates has the blaDHA gene .