CTX-M β-lactamases and associated integrons: their dissemination in Gram-negative bacteria
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Date
29/06/2013Author
Dimude, Juachi Uzochukwu
Metadata
Abstract
Gram-negative bacteria are able to cause many infections including blood stream
infections (BSI).These bacteria may become resistant to antibiotics, often by
acquiring genes in the presence of antibiotic selection pressure. Multi drug resistant
Gram-negative bacteria have become an increasing problem worldwide. A study of
antibiotic resistance in Gram-negative bacteria isolated from blood cultures from
patients in the New Royal Infirmary of Edinburgh (NRIE) was performed. In addition,
a study was performed on isolates from patients in an intensive care unit in Egypt. All
isolates were investigated for susceptibility to an extensive range of antibiotics.
Gram-negative bacteria from Edinburgh found to be resistant to either cefotaxime or
ceftazidime were investigated further. Among the cefotaxime/ceftazidime resistant
isolates, Polymerase Chain Reaction (PCR) analysis revealed the presence of CTX-M-
β-lactamases. Seven E.coli isolates were found to have CTX-M-15 β-lactamases while
the CTX-M-14 β-lactamase was detected in six Enterobacter cloacae. The insertion
sequence ISEcp1 was detected upstream of the blaCTX-M-15 gene in some isolates while
IS26 was found truncating the ISEcp1 in other isolates. Conjugation experiments
found the blaCTX-M-15 gene was transferable to E. coli J62-2. All the isolates had
detectable plasmids, a plasmid ~260kb carried the blaCTX-M-15 gene. Analysis of the
-containing isolates by PFGE shows that those carrying the CTX-M-14
β-lactamase were identical indicating cross infection within the hospital. The CTX-M-
15 β-lactamase-containing isolates showed four isolates had ≥85% similarity but the
others were diverse. Class 1 integrons were found in eight of the CTX-M β-lactamase containing
isolates with the associated gene cassette and sul1 gene.
The isolates from Egypt were found to be resistant to carbapenem, which is the final
mainstream antibiotic option in the treatment of multidrug resistant Gram-negative
bacteria. Further analysis revealed all carried the CTX-M-14 β-lactamase and two
additionally carried the VIM-4 metallo β-lactamase, which accounted for the
resistance to the carbapenems. Furthermore, the insertion sequence ISEcp1 was found
upstream of the blaCTX-M-14 gene in two of the isolates. The blaVIM-4 gene was found to
be part of the gene cassette in the class 1 integron associate with complex ISCR1.
Two of the Egyptian isolates had a detectable plasmid, ~300kb in size, which carried
both blaCTX-M-14 and blaVIM-4 genes.
All the blood culture isolates were examined to ascertain the persistence of
sulphonamide resistance despite the long-term prescribing reduction on this
antibacterial. PCR was performed to detect sul1, sul2 and sul3 genes in all the
isolates. Of the sulphonamide resistant isolates 25 carried the sul1, 27 carried the sul2
and none carried the sul3 genes. Eight isolates had both the sul1 and sul2 genes. Most
of the isolates carried sul1 had Int1 as part of the same class 1 integron. Interestingly
three isolates were PCR negative for sul1 but positive for sul2 and int1. Int2 and 3
were found in 3 and 2 isolates respectively. The class 1 integron contained different
insert gene cassettes; dfrA (dfrA17, dfrA16, dfrA15), aadA (aadA5, aadA2, aadA1)
and blaOXA-1 families in addition to the resident sul gene.
In conclusion this thesis shows the diversity of the genetic environment and carriers
of the CTX-M β-lactamases within the same hospital. Sulphonamide resistance in
Gram-negatives persists despite the prescribing reduction of this antibacterial in a
Scottish hospital and the recommended constraint on the use of sulphonamide.