Isolation and Identification of Pseudomonas spp.
Out of 150 samples collected for bacteriological isolation and identification of
Pseudomonas and
Pseudomonas aeruginosa by phenotypic and genotypic methods, only 10 (6.66%) samples yielded
Pseudomonas. All the 10 isolates producing characteristic colonies with greenish pigmentation, fruity odour on nutrient agar and greenish yellow colonies, sweet odour on Pseudomonas base agar and Gram negative rods were suggestive of
Pseudomonas. The results of biochemical tests were in agreement with the standard book reference and other reports (
Barrow and Feltham, 1993;
Abd El-Tawab et al., 2014,
Shahat et al., 2019).
Ten isolates of
Pseudomonas were further tested and positive amplification of 504 bp fragment specific for the
oprL gene was disclosed only in 7 (70%) isolates and was thus confirmed as
Pseudomonas aeruginosa (Plate 1).
Many researchers have made attempts to develop molecular methods especially PCR for the detection of
P. aeruginosa (
Nikbin et al., 2012)
. In this study the size of amplicon for the gene of interest was 504 bp. The reports which used same set of primer and same PCR amplified fragment size to confirm
Pseudomonas aeruginosa strain genotypically were
Abd El-Tawab et al., (2014);
Elsayed et al., (2016); Bakheet and Torra (2020) and
Soha et al., (2021).
Identification of
P. aeruginosa can be achieved by amplification of a peptidoglycan associated lipoprotein (
oprL) gene (species-specific gene). It encodes a protein in the inner and outer membranes and is essential for the invasion of epithelial cells and indicate also the pathogenic potential of these isolates
(Elsayed et al., 2016).
Detection of antimicrobial resistance genes by PCR in Pseudomonas spp.
Extracted DNAs from 10 isolates of
Pseudomonas spp. were amplified in PCR for detection of various genes of antimicrobial resistance. Different primers used for targeted antibiotic genes were
blaCTX-M,
tetA,
MexA,
Sul1,
blaSHV
, blaTEM and
tetB (Table 1). Amplification and detection of
blaCTX-M gene was done using published primer as per
Adesoji et al., (2015). An amplicon size of 538 bp was taken as positive for
blaCTX-M gene. Out of 10, one (10%) isolate was positive for
blaCTX-M gene (Plate 2). A desired product of 628 bp using primer as per
Gundran et al., (2020) in PCR was produced by 2 (20%), strains thus showing the presence of
blaSHV gene against beta lactam antibiotics (Plate 3). Amplification and detection of
blaTEM gene was done using published primer as per
Gundran et al., (2020). An amplicon size of 506 bp was taken as positive for
blaTEM gene. Out of 10 isolates, 5 (50%) were positive for
blaTEM gene (Plate 4). The detection of ESBL and beta-lactamase encoding genes:
blaTEM,
blaSHV and
blaCTX in the ESBL-producing
Pseudomonas species isolated from livestock samples were also reported by
Falodun et al., (2020).
In this study the ESBL producing
Pseudomonas isolates were dominated by encoding
blaTEM gene, followed by
blaSHV and
blaCTX-Ms (Table 3). It has been reported that ESBL genes show variation depending on the geographical location. Dominance of genotype
blaTEM was also reported by
Chen (2015). On contrary,
Jamali et al., (2017) reported the prevalent gene to be
blaSHV. The least detected ESBL genotype from this study was
blaCTX-M similar to records of
Miranda (2015). In other studies
Hassan et al., (2020) reported that resistance genes,
blaCTX
, was detected in 100% of the examined
P. aeruginosa isolates although the lower prevalence was recorded by others
(Peymani et al., 2017). The detection of
blaCTX gene among all the tested isolates could explain the growing resistance pattern of
P. aeruginosa, which reached to complete resistance against the third generation of cephalosporins represented by cefotaxime.
Several researchers have reported about the concurrence of different β-lactamase genes found in the same strains
(Bahrami et al., 2018). ESBL combinations in our study observed were
blaTEM +
blaSHV and
blaTEM +
blaSHV+
blaCTX-M.
Chen (2015) reported the commonest combination of genes to be
blaSHV +
blaCTX-M. Prior to now,
blaTEM used to be the most prevalent gene but recent reports suggest that the CTX-M-type group of ESBLs may now be the most predominant type globally
(Sahoo et al., 2019). These discrepancies may be due in part to varied geographic location, different levels of healthcare facilities involved, varied levels of exposure to healthcare settings, antibiotic use and antibiotic stewardship practices.
ESBLs are undergoing continuous mutations, causing the progression of new enzymes, showing expanded substrate profiles. So far, there have been more than 300 different ESBL variants which have been clustered into nine different structural and evolutionary families based on their amino acid sequences
(Bokaeian et al., 2015).
A desired product of 316 bp using published primer as per
Farghaly et al., (2017) in PCR was produced by 9 (90%), strains thus showing the presence of
MexA gene against beta lactam antibiotics (Plate 5). Detection of some efflux pumps system genes, such as
oprJ, mexA, etc in a
vian
P. aeruginosa isolates was conducted by
Farghaly et al., (2017) and the results revealed the presence of these genes with percentages of 100% and 85.7%, respectively.
The DNA extracted from 10 isolates of
Pseudomonas was subjected to PCR for detection of
Sul1 gene using published primer pair
(Shehata et al., 2016). A desired amplicon size of 822 bp was found in only 3 (30%) out of 10
Pseudomonas isolates (Plate 6).
Adekanmbi et al., (2020) also reported
Sul1 gene in
P. aeruginosa. Resistance to sulfonamides occurs principally through the acquisition of the alternative dihydropteroate synthase (DHPS) gene
sul, the product of which has a low affinity for sulfonamides
(Changkaew et al., 2014).
Three (30%) out of 10
Pseudomonas strains showed the presence of
tetA gene against tetracycline group of antibiotics, as they produced a desired product of 494 bp in PCR as per
Adesoji et al., (2015) (Plate 7). Two (30%) out of 10
Pseudomonas strains showed the presence of
tetB gene against tetracycline group of antibiotics, as they produced a desired product of 773 bp in PCR as per
Van et al., (2008) (Plate 8). One of the most common resistance mechanisms in Gram-negative bacteria is the energy-dependent efflux pump system which is encoded by the genes
tetA,
tetB,
tetC,
tetD and
tetG, with
tetA and
tetB genes being the most frequently described
(Lewis et al., 2002).
Prevalence of different antimicrobial genes in Pseudomonas isolates
Seven antimicrobial resistant genes (ARGs), belonging to three antimicrobial families, were detected in varying percentage from all the
Pseudomonas isolates of different samples as shown in Table 3. Analysis revealed that the most prevalent gene found in
Pseudomonas isolates was
MexA detected in 9 (90%) isolates followed by
blaTEM (50%),
tetA and
Sul1 (30%),
tetB and
blaSHV (20%) and
blaCTX-M (10%) genes (Fig 1). Maximum 7 genes detected in one sample followed by 5, 4, 2 and 1 in other samples (Fig 2). MexA gene was detected in maximum 9 isolates and
blaCTX-M in minimum one isolate (Table 3).
Analysis revealed that the most prevalent gene found in
Pseudomonas isolates was
MexA detected in 9 (90%) isolates followed by
blaTEM (50%),
tetA and
Sul1 (30%),
tetB and
blaSHV (20%) and
blaCTX-M (10%) genes (Fig1). Maximum 7 genes detected in one sample followed by 5, 4, 2 and 1 in other samples (Fig 1). MexA gene was detected in maximum 9 isolates and
blaCTX-M in minimum one isolate (Table 3).
Gene sequencing
For the sequence analysis, the PCR product of two genes (
oprL and
MexA) were got sequenced from Eurofins. Nucleotide base analysis of all sequence in GeneBank database revealed 98-100% homology with
Pseudomonas aeruginosa.
