The study investigated the frequency of antibiotic resistance and ESBL producers in healthy poultry and also to determine the transferability and potential exchange of ESBL producing
E. coli. After initial screening of samples ESBL
E. coli was isolated from 135 samples and 265 samples were negative, yielding an isolation rate of 33.75 per cent regardless of the area and poultry sale outlet (Fig 1).
A comparative study observed maximum per cent isolation with Combined Disc DiffusionTest (CDDT) method (Table 2; Fig 2, 3) which means sample resistant to cefotaxime, cefpodoxime and few towards ceftazidime. European Union also recommends resistance of ESBL producers to cefotaxime, variably resistant to ceftazidime and susceptible to cefoxitin. (
European Food Safety Authority, 2011d;
European Food Safety Authority, 2011b), in reference to the above recommendations cefotaxime was included as marker for present investigations. (
European Food Safety Authority, 2012;
European Food Safety Authority, 2011b). Taslima, (2012) and
George et al., (2005) even recommended Combined Disc DiffusionTest (CDDT) as the single best method for ESBL
E.coli isolation. Many times strip cannot be read due to growth beyond the MIC range of the strip, make it less sensitive for detection
(Drieux et al., 2008; Garec et al., 2011).
Sample analyzed (Table 3) The study by multiplex pcr
(Monstein et al., 2007; Pierano and Pitout,2010) for
blaTEM
blaSHV and
blaCTX genes revealed 64.47 per cent
blaTEM, 19.73
blaCTX and 2.63 per cent
blaSHV genes 15.78 both
blaCTX and
blaSHV genes and 1.31 per cent blaTEM and
blaSHV genes from phenotypically confirmed ESBL producing
E. coli isolates. It correlates with the findings of 14 different chicken farms in Henan Province in China where 80.7 per cent isolates harboured
blaTEM genes and 51 per cent isolates carried
blaCTX-M genes
(Yuan et al., 2009). In India too TEM and CTX were (39.2 per cent) predominantly found in
E.coli. Presence of both
blaTEM
blaSHV genes in a sample suggest, same organism can harbour both TEM and SHV type beta lactamase
(Yan et al., 2000) (Table 3). As the PCR-based results matched with the laboratory-grown cultures, it is concluded that direct diagnosis of
E. coli and/or other species of bacteria by PCR is possible directly from swab samples. It also simulate with the study undertaken by
Shailash et al., (2010), where sixteen strains of
E. coli were successfully isolated from biopsy/swab samples of 15 out of 42 patients (35.71 per cent) admitted to the S.S Hospital, Varanasi. Two
E. coli strains were isolated from Diabetic foot ulcer of one patient (DF30). Identity of
E. coli strains was confirmed by amplification of the
E. coli specific 16S rDNA.
The early ESBL’s evolved from TEM assumed to have an origin in
E.coli (
Witte, 1998). Beta lactamase SHV type, first detected in Germany is assumed to have transferred horizontally to other bacteria including
E.coli. Since not much information is available about the prevalence of gene responsible for ESBL production in
E.coli in poultry in India, it is assumed that high rate of ESBL isolation by phenotypic method may be due to mutation of any gene (TEM or SHV) and some newer prevalent gene apart from CTX
(Yan et al., 2000).
Positive Isolates were later subjected to study inhibitory potential, per cent inhibition and antibacterial potentiation of
Syzygium aromaticum, Punica granatum against extended spectrum beta lactamase enzyme by colorimetric method (microplate assay method) using CENTA and Nitrocefin as the chromogenic substrate at the wave length of 405 and 486 nm respectively. Oil of
Syzygium aromaticum showed maximium per cent inhibition and minimum absorbance value (0.4±0.02, 28 per cent and 0.41±0.03, 27) (p>0.05) with no significant difference with CENTA and NITROCEFIN (Table 4 to Table 7).
Punica granatum depicted mean absorbance value and per cent inhibition of 1.72±0.05, 14.0 and 1.73±0.05,13.9 and with CENTA and NITROCEFIN respectively (Table 5). Combination of oil of
Syzygium aromaticum and
Punica granatum showed 1.41±0.04, 31.9 per cent and 1.38±0.04, 31 per cent inhibition (Table 6). Tazobactum, (100µM) taken as the standard control exhibited 0.12±0.01 and 0.13±0.01 (Mean±S.E.) of inhibitory potential, per cent inhibition observed was 99.88 per cent and 98 per cent.
Lena et al., (2013), also observed cinnamon and clove oil exhibited stronger antibacterial activity against these ESBL isolates than Tulsi, Garlic or Neem oil. Approximately 86 per cent and 100 per cent of
E. coli and 100 per cent of
K. pneumoniae were sensitive to eugenol and cinnamaldehyde (active principle of clove oil), respectively.
According to (
Mishra and Mishra 2010) level of antibacterial effect of
Ocimum sanctum against
E.coli was found to be lower as compared with the other herbs specially clove oil which also simulate with the present investigation, where effect of
Punica granatum was fairly less than the
Syzigium aromaticum.
