Phenotypic and genotypic detection of antibiotic resistance of Pseudomonas aeruginosa isolated from urinary tract infections

Bakground Pseudomonas aeruginosa is a major nosocomial uropathogen. It can tolerate a wide variety of physical conditions and many antibiotics by different resistance mechanisms. Objectives This study aimed to investigate the mechanisms of antibiotics resistance in uropathogenic P. aeruginosa clinical isolates. Methods Two hundred sixty six urine samples were collected from Zagazig University Hospitals, Zagazig, Egypt. P. aeruginosa isolates were identified using standard microbiological tests. The sensitivity to different antibiotics was determined by disc diffusion method. Anti-microbial resistance mechanisms were investigated using phenotypic methods and confirmed by PCR. Results Fifty P. aeruginosa isolates were recovered. All isolates were MDR and were resistant to amoxicillin/clavulinic, sulphamethaxzole/trimethoprim, doxycycline and ceftazidime. Phenotypic detection of resistance mechanisms revealed that all strains have efflux mechanism, outer membrane porins, and AmpC β-lactamase; none of the strains showed ESBL activity and two of the imipenem resistant strains showed MβL activity. PCR analysis showed that all strains have MexAB-R, OprD and AmpC genes, 42 strains had PSE gene, while VEB and VIM genes were not detected. Conclusion The resistance rates in P. aeruginosa were higher than global values; this resistance was attributed to several mechanisms. This high resistance is alarming and necessitates applying strict antibiotic prescription policies.


Introduction
Pseudomonas aeruginosa is one of the most ecologically significant species among the genus Pseudomonas. P. aeruginosa is of extreme importance because of the widespread distribution of its strains in nature, its high intrinsic anti-bacterial resistance and its virulence 1 .
Bacterial efflux pumps are greatly involved in the intrinsic resistance of Gram-negative bacteria. When overexpressed, efflux pumps can confer high resistance to previously effective antibiotics. Many efflux pumps transport a wide range of unrelated drugs and are known as multidrug resistance (MDR) efflux pumps. Four antibiotic efflux systems have been reported in P. aeruginosa. Mex-AB-OprM is the efflux system that is responsible for extrusion of β-lactams and quinolones 15 .
The outer membrane of P. aeruginosa represents a significant barrier that hinders the penetration of antibiotics. β-lactams and quinolones can only cross the outer membrane through the porin proteins. P. aeruginosa produces several porins such as OprD and OprF. Loss or diminished expression of OprD are frequently related to mipenem resistance i 16 .
This study was performed to detect antibiotic resistance profile of local P. aeruginosa isolated from urinary tract infections and to determine the underlying resistance mechanisms by phenotypic and genotypic methods.

Material and methods Media and chemicals
Mueller Hinton broth, Mueller Hinton agar and antibiotic discs were purchased from Oxoid (Hampshire, UK). Ethidium bromide was obtained from Merck, Hohen-burnn, Germany. MyTaqTM master mix was the product of Bioline Reagents Limited, UK, while Gene-Ruler 100 bp DNA Ladder was purchased from Thermoscientific Inc, USA. Other chemicals were of pharmaceutical grade.

Bacterial strains
A total of 266 urine samples were collected from patients with urinary tract infections admitted to Urology Department, Zagazig University hospitals, Zagazig, Egypt. Only one sample was collected per patient. Samples were collected from clean-catch midstream fresh urine in sterile plastic jars. Samples were immediately transported to the Microbiological laboratory at Faculty of Pharmacy, Zagazig University, where they were immediately processed. P. aeruginosa identification was based on standard microbiological technique including: Gram staining, colony morphology, motility, pigment production, oxidase reaction, growth at 42 °C, gelatin liquefaction test and sugar utilization tests 17 .

Phenotypic detection of resistance mechanisms: Phenotypic detection of MβL activity:
The imipenem-resistant P. aeruginosa isolates were investigated for MβL production by imipenem-Ethylene diamine tetra acetic acid combined disc test (IMP-EDTA CDT) as described previously by Yong et al. 19 Briefly, overnight culture of the test organism was prepared and its turbidity was adjusted to a 0.5 McFarland standard and surface inoculated on MHA plate (9 cm in diameter). Two imipenem discs (10 μg) were placed at a distance of 4-5 cm from each other on the plate, and amount of 10 μL of 0.5M EDTA solution was added to one of them. The inhibition zones of the imipenem and imipenem-EDTA discs were compared after 18 h of incubation at 35 °C. Isolates were considered as MβL positive if the zone diameter of imipenem-EDTA disc was larger by more than or equal to 7 mm.

Phenotypic detection of AmpC β-lactamases production:
AmpC β-lactamase production was phenotypically detected on the isolates that were resistant to cefoxitin according to Vanwynsberghe et al. 20 Briefly, ceftazidime (30 μg) and cefotaxime (30 μg) disks were each placed at a distance of 20 mm from cefoxitin (30 μg) disk on a 9 cm-diameter MHA plate inoculated with the test P. aeruginosa isolate. The production of AmpC enzyme was confirmed when the zones of inhibition produced by either of the cephalosporins (ceftazidime or cefotaxime) when used in conjunction with cefoxitin were ≥5 mm larger than cephalosporins inhibition zone alone.

Phenotypic detection of extended spectrum β-lactamases (ESBL) production:
A lawn culture of the organisms was made on a 9 cm-diameter MHA plate, as recommended by CLSI 21 . A disc which contained ceftazidime-clavulanate (30/10 µg) and ceftazidime discs (30 μg) were placed at distance of 20 mm. After an overnight incubation at 37°C, an increase of 5 mm in zone of inhibition of the combination discs in comparison to the ceftazidime disc alone was considered to be ESBL producer.

Phenotypic detection of efflux pumps by ethidium bromide cartwheel (EtBr-CW) method
The ability of efflux pumps to expel ethidium bromide was assessed according to EtBr-CW method 22 . Trypticase Soya Agar (TSA) plates containing EtBr ranging from 0 to 4 mg/L (these concentrations were determined according to the bacterial MICs of EtBr) were freshly prepared on the same day of the experiment and kept protected from light. Overnight cultures of the tested bacterial isolates were prepared and adjusted to a 0.5 McFarland turbidity standard. The 9 cm-diameter TSA plates were divided into ten to twelve sectors forming a cartwheel pattern.
The adjusted bacterial cultures were swabbed on the Et-Br-TSA plates starting from the center of the plate to the margin. After incubation of the plates at 37 ºC for 16 h, the plates were examined under UV transilluminator (Cole-parmer, Vemon Hills, USA), the minimum concentration of EtBr that produced fluorescence of the bacterial mass was recorded. The isolates were considered Et-Br-CW-negative if they showed emission of fluorescence at 0.5-1 mg/L EtBr, EtBr-CW intermediate (emitting fluorescence at 2 mg/L) or EtBrCW-positive (emitting fluorescence only at 3-4 mg/L).

Phenotypic detection of outer membrane permeability (OMP)
Assessment of MDR bacteria for OMP was performed by determination of the minimum inhibitory concentration (MIC) values for selected antibiotics in the presence and absence of EDTA (pH=7.2); a permeabilizer which chelates divalent cations that stabilize molecular interactions in the OM causing disruption of OMP. To avoid the effect of EDTA on bacterial growth, it was used at a concentration of ¼ MIC. A four-fold reduction in the antibiotic MIC or more in the presence of EDTA indicates OM reduced permeability activity.
The MICs of antibiotics and EDTA were determined by broth micro-dilution according to CLSI guidelines 23 . Müller Hinton broth (MHB) was inoculated with colonies of P. aeruginosa and broth was incubated with shaking at 37°C until the turbidity became equivalent to 0.5 McFarland standard. Then the bacterial suspension was diluted 1:100 in MHB medium. Two-fold serial dilutions of each drug were prepared in 96 wells microtiter plates in a final volume of 100 µL per well. Each well was inoculated with 100 µL of the previously prepared bacterial suspension and incubated at 37°C for 18 -20 h. MIC was defined as the lowest concentration of drug at which there was no visible growth of the organism. The results were recorded and interpreted according to CLSI 23 .

Genotypic detection of resistance mechanisms by PCR.
The gDNA was extracted by picking a colony from an agar plate using a sterile pipette tip and resuspending it into 20 μL of distilled water. The mixture was vortexed for 10 s then incubated at 98 °C for 5 min. The lysate was African Health Sciences Vol 18 Issue 1, March, 2018 centrifuged and the resulting supernatant was collected, diluted with distilled water at a 1:3 dilution ratio, and subjected to PCR analysis. Each PCR mixture contained 10 µL of MyTaqTM master mix; 1.5 µL of forward primer, 1.5 µL of reverse primer, 2 µL of gDNA template and nuclease free water to 20 µL. The primers used in this study are listed in Table 1. The PCR was performed in Biometra T-personal thermocycler (Goettingen, Germany). The PCR products as well as Gene-Ruler 100 bp DNA Ladder were separated on 1% agarose gel, stained with 2 µL of EtBr, and visualized by UV transilluminator and photographed.
Detection of Mex-ABR genes was done according to Al-Grawi et al. 24 The cycling conditions were initial heating at 94°C for 3 min, then 32 cycles of 94°C for 30 sec, 57°C for 45 sec and 72°C for 1min and final extension at 72°C for 7min. OprD gene detection was performed according to Rodríguez-Martínez et al. 25

Antimicrobial susceptibility
Only 50 P. aeruginosa strains were recovered from the 266 urine samples (18.7%). The resistance rates of P. aeruginosa isolates to the tested antibiotics were presented in Figure 1.

Phenotypic detection of resistance mechanisms
The IMP-EDTA-CDT was performed for the 5 imipenem resistant isolates and the results revealed that 2 of these isolates were MβL producer (Fig. 2 A). All the 50 MDR P. aeruginosa isolates were phenotypically confirmed to produce AmpC β-lactamase (Fig. 2 B). All the MDR isolates were subjected to qualitative assessment for ef-flux pumps by EtBr-CW method, and all of the tested isolates showed efflux pump activity (Fig. 2 C). None of the MDR P. aeruginosa isolates were ESBL producers (Fig.  2 D). EDTA was used for phenotypic detection of OMP; generally EDTA potentiated the effect of different antimicrobial agents as it reduced the tested antibiotics MICs as indicated in Table 3.  Genotypic detection of resistance genes PCR was used to detect resistance genes. Multiplex PCR was used for amplification of MexA-B-R genes encoding efflux pumps, all the MDR isolates gave a triple band at 280, 411 and 503 bp matching the Mex-B-R-A genes (Fig.  3 A).
For OprD gene encoding outer membrane porins, all of the MDR isolates gave a single band at 242 bp match-ing OprD gene (Fig. 3 B). For amplification of AmpC gene encoding AmpC β-lactamase, all the MDR isolates gave a single band at 124 bp matching the AmpC gene (Fig. 3 C). In case of PSE gene, 42 of the MDR isolates gave a single band at 698 bp matching the PSE gene (Fig.  3 D). For VEB gene encoding ESBL enzyme and VIM gene encoding MβL, none of the tested isolates gave the 648 bp band matched to VEB gene or the 390 bp band matching VIB gene.

Discussion
MDR isolates represents a prominent health problem in Egypt due to several factors including misuse of antibiotics and biocides 30 . In the current study, 50 P. aeruginosa isolates were recovered from urine samples, all the isolates were MDR (100%), this MDR rate was higher than rates reported previously in Egypt which ranged from 34% -56% [31][32][33] ; this may reflect higher resistance rate in case of urinary tract infections. Other global studies showed even lower MDR rates; 5.9% in Canada 34 , 19% in Germany 35 , 19.6 % in Malaysia 36 and 20.7% in Nepal 37 . This MDR rate is alarming and necessities application of strict antibiotic prescription policies in our country.
In the current study, phenotypic detection of MβL production reveled that only 2 of the 5 imipenem resistant isolates (40%) were MβL-positive. A previous Egyptian study reported relatively similar results where 27% of P. aeruginosa were MβL producers 39 . PCR analysis showed that VIM gene (encoding MβL) was not detected in any of our isolates. High prevalence of VIM gene (58-61%) was reported previously in P. aeruginosa 40 , Probably the MβL activity in our study is attributed to other MβL genes or another mechanism. Several mechanisms of imipenem resistance have been reported previously including secretion of carbapenemases, increased expression of efflux systems, and reduced porin expression 41 . The isolation of carbapenem resistant strains is alarming and requires restriction on prescription of these valuable drugs Phenotypic analysis showed that none of our isolates was an ESBL producer, this was comparable to the low occur-rence (7.4%) reported previously in Egypt 40 . While Lin et al. 42 found that 29% of his strains were ESBL producers. PCR was used for detection of VEB gene encoding ESBLs, the gene was not detected in any of the isolate which is compatible with our phenotypic results. Zafer et al. 40 reported that 10.4% of ESBL-producing P. aeruginosa isolates were positive for VEB gene.
All of the tested isolates were AmpC β-lactamase producer. Chika et al. 43 found that 36% of P. aeruginosa isolates were AmpC producers. The presence of AmpC -lactamase enzyme was confirmed by PCR, all of the P. aeruginosa isolates harbored the AmpC gene. Similarly, Fazeli et al. 26 found that all the P. aeruginosa isolates had AmpC gene.
The Pseudomonas specific enzyme (PSE) belong to Class A carbenicillin hydrolysing β-lactamases that confer resistance to carbenicillin and pipracillin. The presence of PSE gene was tested by PCR, 42 isolate (84%) were found to have the PSE gene, it is worth mentioning that the 8 PSE-negative isolates were susceptible to pipracillin. Cho et al. 44 reported lower rate where only two out of 61 Pseudomonas isolates (3.3%) harbored PSE gene. The AmpC and PSE are the main hydrolyzing enzymes that confer β-lactam resistance in our study.
The efflux system contributes to the natural bacterial resistance to a wide range of antibiotics and detergents 45,46 . In our study, active efflux was detected in all isolates. This was in accordance with Rana et al. 47 who reported presence of active efflux in all MDR isolates. Presence of MexABR efflux system was confirmed by amplification of this operon using multiplex PCR. All of the P. aeruginosa isolates were positive to the three tested genes. This was in accordance with Al-Grawi et al. 24 who found that all of the P. aeruginosa isolates were positive to the three genes.
Our study also investigated the outer membrane reduced permeability as a resistance mechanism; assay was performed with EDTA as a permeabilizer. The MIC values for tested antibiotics was determined in the presence and absence of sub MIC (1/4 MIC) of EDTA. Our results showed that EDTA has augmenting effect on different anti-microbial agents; EDTA reduced the MICs of various anti-microbials by up to 16 folds. This was compa-rable with the results of Ayres et al. 48 who reported that EDTA was greatly enhanced the activity of various antibiotics and biocides against Gram negative isolates.

Conclusion
This study revealed that the resistance rates of local P. aeruginosa were higher than worldwide values. This high resistance was attributed to several mechanisms including efflux pumps, reduced activity of outer membrane porins, production of AmpC and PSE β-lactamases. This high MDR resistance rate is alarming which necessitates applying strict antibiotic usage and prescription policies. Also selection of curative antibiotics should depend on the anti-microbial susceptibility results.