PHENOTYPIC AND MOLECULARCHARACTERIZATION OF PLASMID -MEDIATED AMPC β- LACTAMASES AMONG GRAMNEGATIVE CLINICAL ISOLATES

Amira Hamed Afify. There are currently no standardized phenotypic methods for the screening and detection of plasmid -mediated AmpC enzymes. Aim: to evaluate two phenotypic methods (AmpC E test and cefoxitin – cloxacillin double disc synergy) to detect AmpC enzymes in Escherichia coli, Klebsiella spp., and Proteus mirabilis using multiplex PCR as gold standard method. Materials and methods: total of 1500 gram negative isolates were screened for potential plasmid-mediated AmpC enzymes by Cefoxitindisc.AmpC E test and cefoxitin –cloxacillin double disc synergy tests were used to confirm detection of plasmid-mediated AmpC enzymes.The genotypic identification was done using multiplex PCR. Results:The potential Amp C-producing isolates among all the studied isolates were only 4.7 % (70/1500) by cefoxitin disc. Among the cefoxitin resistant isolates,22.9 % and 24.3 % confirmed to be P-AmpC by cefoxitincloxacillin double disc synergy test and AmpC-Etest, respectively. Plasmid encoded AmpC genes were detected by PCR in 27% of cefoxitin resistant isolates. The most prevalent AmpC gene family was CIT and MOX.The sensitivity of AmpC E test and cefoxitin –cloxacillin double disc synergy were 81.3 % and100 % respectively and the specificity were 92.3% and 95.9%.

PAmpC beta-lactamases are class C or group I cephalosporinases that confer resistance to a wide variety of β-lactam antibiotics including penicillins, expanded-spectrum cephalosporins (with the exception of cefepime and cefpirome), cephamycins, monobactams, and beta-lactam inhibitors. In contrast to expanded-spectrum betalactamases (ESBLs), AmpC beta-lactamases are inhibited by boronic acid and cloxacillin (Tan et al, 2009). Currently there are no CLSI guidelines for pAmpC detection. Reduced susceptibility to cefoxitin in the Enterobacteriaceae may be an indicator of AmpC activity; however cefoxitin resistance may be mediated by mechanisms other than AmpC such as porin channel mutation Hence it should be confirmed by other tests ( Ananthan and Subha, 2005

Materials and methods:-
Bacterial Isolates:-A total of 1500 nonduplicate clinical isolates that lack or minimally expressing chromosomally encoded AmpC beta-lactamases; including E. coli=768, K. Pneumoniae = 662 and Proteus mirabilis=42) recovered from the microbiology lab of Zagazig University hospital during the time period from July 2014 to February 2015.The clinical isolates were collected from different clinical samples (Pus, sputum, blood, urine, CSF and other body fluids). All the isolates were identified by MALI-TOF mass spectrometry.

Screening for AmpC Production:-
The clinical isolates were screened for cefoxitin (30 μg) insusceptibility as an indicator for AmpC production by Kirby Bauer disc diffusion test disc. The inhibition zone sizes were interpreted according to CLSI guidelines. Isolates with inhibition zone diameter ≤18 mm were considered potential Amp C producers.

Phenotypic Tests for Detection of AmpCβ-lactamases:-
All the screen positive isolates were subjected to two confirmatory phenotypic tests (AmpC E test and cefoxitincloxacillin double disc synergy). Klebsiella Pneumonia ATCC-1144ᵀᴹ (Microbiologics, MediMark, Europe) was used as Positive control strain for PAmpC.
The Etest AmpC (BioMérieux SA, France):-E test was performed according to the manufacturer's instructions. The test principle comprises a strip impregnated with a concentration gradient of cefotetan on one half of the strip and cefotetan with cloxacillin on the other half of the strip. MICs of cefotetan alone and cefotetan with cloxacillin were determined as recommended by the manufacturer. Ratios of cefotetan versus cefotetan/cloxacillin of ≥8 were considered positive for AmpC betalactamase production.

The cefoxitin-cloxacillin double disc synergy test (CC-DDS) (HI Media Laboratories Pvt.Ltd):-
This test is based on the inhibitory effect of cloxacillin on AmpC using disks containing either 30 μg of cefoxitin or 30 μg of cefoxitin plus 200 μg of cloxacillin. A difference in the cefoxitin-cloxacillin inhibition zones minus the cefoxitin alone zones of ≥4 mm was considered indicative for AmpC production.

Detection of bla PampC by multiplex PCR:-
Bacterial DNA was extracted using PureLink Genomic® DNA Mini kit-Invitrogen-life technologies according to the manufacturer's instructions. Genes encoding PMABLs were amplified using the primers described by Perez-Perez &Hanson (2002).All primers were synthesized and supplied by Invitrogen-life technologies.
PCR was performed using the following conditions: initialdenaturation step at 95˚C for 5 min followed by 30 cycles of Denaturation at 94˚C for 45sec, Annealing at 62˚Cfor 45 sec, Extension at 72˚C for 1 min, with final extension at 72˚C for 5 min. PCR products were visualized on a 2 % agarose gel stained with ethidium bromide.
Statistical analysis:-All data were coded, checked, entered and analysed using SPSS(statistical package for social science) software version 18;Performance of phenotypic tests is assessed by sensitivity, specificity, positive predictive value, negative predictive value and accuracy.

Comparison of confirmation assays for AmpC production:-
The performance of Etest AmpC and the cefoxitin-cloxacillin CC-DDS method were compared as phenotypic confirmation tests using multiplex PCR as the gold standard method. The results of the Etest AmpC were inconclusive in 3 isolates where MICs exceeded the scale of the test for cefotetan alone and cefotetan in combination with cloxacillin. With the CC-DDS, 7 inconclusive results were observed where no inhibition zone was present for cefoxitin alone and in combination with cloxacillin. Isolates with inconclusive results were not included in the calculation of performance parameters.   From the distribution of different P AmpC β-lactamase genes among the clinical isolates (Table3), it was observed that enzymes from the AmpC group were predominantly present in k.pneumonae isolates (28, 6%) followed by E.coli (23.9%)

Conclusion:-
Isolates of E. coli, K. pneumoniae, and Proteus mirabilis showed the occurrence of plasmid mediated AmpC βlactamase which is alarmingbecause of probability of dissemination of these plasmid mediated resistance genes within the hospital.The multiplex PCR revealed that CIT and MOX are the most predominant genes.Regarding phenotypic confirmatory tests, CC-DDS showed higher sensitivity and specificity than Etest AmpC, besides E-test AmpC is costly compared to CC-DDS to be used by clinical laboratories for routine PAmpC screening procedureTherefore, CC-DDS is a more suitable routine confirmatory procedure for early detection of PAmpCproducing bacteria.