Genotyping Investigation of Ciprofloxacin Resistance Genes (Parc And Pare) In Clinical Pseudomonas Aeruginosa Isolates

doi:10.21203/rs.3.rs-2742082/v1

Purpose

Due to Pseudomonas aeruginosa's low susceptibility to the development of antibiotic resistance and antimicrobial drugs, therapy and control of severe infections often result in recurrent difficulties. A bacterial isolate with multidrug resistance (MDR) is one that is resistant to at least one compound from more than three antimicrobial groups, such as aminoglycosides and fluoroquinolones (10). Human burn wounds are complex environments where necrotic tissue and exudates formed from plasma develop.

Methods

The samples were immediately cultivated on Pseudomonas aeruginosa-selective Blood, MacConkey, Nutrient, and Cetrimide agar media and incubated overnight at 37°C under aerobic conditions. Several biochemical tests, including Oxidase and Catalase tests, were used to identify P. aeruginosa. Additionally, the identification was confirmed using a quick identification procedure (VITEK 2 System). After adding 20% glycerol, the detected isolates were kept at -20°C.

Results

The findings of the local isolates' sequences being aligned with the reference strain revealed some mismatches in the nucleotides of our query at various locations, including deletions and replacements. According to the findings in table 4, there were discrepancies between the isolates from burn that were resistant to ciprofloxacin (P1 and P16) and the reference bacteria (CP034435).

Conclusion

The following are the findings of the current study: Pseudomonas aeruginosa is more prevalent in burn samples than in other clinical samples. Misuse of these antibiotics may contribute to an increase in MDR Pseudomonas aeruginosa, particularly resistance to fluroquinolones (Ciprofloxacin).

Pseudomonas aeruginosa

Fluroquinolone

Ciprofloxacin resistance

parC

parE

Pseudomonas aeruginosa appeared with burn patients are considered as one the most important opportunistic pathogen; it causes an extensive range of acute and chronic nosocomial infections. This kind of microorganism can be found in approximately 33% of all burn wounds while in 59% of extensive burns (22). Pseudomonas aeruginosa results recurring complications during treatment and control of severe infections because of the limited susceptibility to the emergence of antibiotic resistance and antimicrobial agents. The Multidrug-Resistance (MDR) is defined as the bacterial isolate which is non-susceptible to at least one agent in greater than three antimicrobial categories, such as Aminoglycosides and Fluoroquinolones (10).Human burn wound is a complicated environment which forms a necrotic tissue and plasma derived exudate.Recently, it is reported that in Burn Wound Exudates (BWE) and compared to other burn wound pathogens, such as Staphylococcusaureus and Escherichiacoli, clinical and laboratory Pseudomonas aeruginosa strains are able to be developed. It is observed that during growing in these environments, the production of Pseudomonas aeruginosa stains of virulence factors, including cytotoxic pigment pyocyanin, secreted elastase and rhamnolipids surfactants is strongly increased (18). One of the major classes of antibiotics which are utilized in the treatment of infections is Fluoroquinolones (FQ), where the treatment of infections is caused by Pseudomonas aeruginosa. Moreover, an increasing rate of FQ resistance among clinical isolates of Pseudomonas aeruginosa has been reported by Large-scale surveillance studies (2). In clinical medicine, Quinolones are vastly utilized in synthetic antimicrobials, because of the extensive use. Theresistance to this antibiotic has emerged, and it will be common in many in numerous bacterial species. In both developing and developed countries, drug resistance is an emerging issue in terms of mortality, morbidity. Since the 1990s, quinolone resistance has first emerged against methicillin-resistant Staphylococcusaureus and Pseudomonasspecies. Quinolone acts by restraining bacterial DNA replication by blocking DNA gyrase/topoisomerase IV enzyme activity. Mutation altering the drug targets (most common) and plasmids coding for defensive proteins can protect the bacteria from the deadly impacts of quinolones. The genes coding for DNA gyrase are gyrA and gyrB and topoisomerase IV are parC and parE(33). Studies have linked to misusing the antibiotic and non-compliance to treatment as the major cause of antibiotic resistance. Moreover, the organisms have devised a strategy to transfer the hereditary material for drug resistance both via chromosomes and plasmids, which is responsible for the fast spread of antibiotic resistance. Higher rates of resistance to quinolones have been detailed from distinctive parts of the world. However, studies on the resistance design to quinolones in this locale are constrained (38). This study detected the predominance of the Ciprofloxacin resistance design within the clinical isolates from this locale by phenotypic and molecular methods using Polymerase Chain Reaction (PCR) and/or Sanger's sequencing to screen Fluoroquinolones region determination resistance (FRDR).

Specimens collection and pathogens Isolation

One hundred and twenty samples were collected during November/2019 to March/2020 from different units of five hospitals in Baghdad Medical city (Ghazi-Al Hariri surgical specialities hospital and specialized Burns Hospital), Al-Kindi Al Karama Teaching Hospital and Nu’man Teaching Hospital. All swabs and specimens were transported to the laboratory under suitable condition. Burn swabs were taken from the deepest burnt tissue via moisten swab with Brain-Heart Infusion broth. The samples were immediately cultured on Blood agar, MacConkey agar, Nutrient agar and Cetrimide agar selective media for Pseudomonas aeruginosa and incubated overnight at 37°C under aerobic conditions. P. aeruginosa was determined using various biochemical tests, including Oxidase test and Catalase tests. Furthermore, rapid identification method (VITEK 2 System) was used to confirm the identification. The identified isolates were stored at -20°C after adding 20% glycerol.

Antimicrobial Susceptibility: All 46 P.aeruginosa isolates from burn samples were subjected to the antibiotic susceptibility test against specific antibiotics. VITEK 2 system was used to achieve this screening by gram-negative susceptibility software (version 5.01 and AST-GN222 cards) following the manufacturer's instructions. The results of the antibiotic susceptibility test were divided into susceptible isolates (S), intermediate isolates (I) and resistant (R) isolates. According to the list of antibiotics presented in the VITEK 2 cassette (AST-GN222), there are four categories used, including Fluoroquinolones (Aminoglycoside (Gentamicin); Carbapenems (Imipenem) and Cephalosporins: (Ceftazidime). The antimicrobial analysis conducted in two replicates.

Molecular investigation

According to antibiotic profile 21 of multidrug resistance (MDR) P. aeruginosa isolates were chosen for Genomic DNA extraction from the culture of using a Genomic DNA kit (Geneaid, Korea)following g the instruction of the manufacturing company. The extracted DNA has been subjected to detect the gyrB gene. The PCR technique has been used for molecular detection using master mix (GoTaq-Green) and specific primers for the gyrB gene with product size of 510bp

F-GCGGTGGAACAGGAGATGGGCAAGTAC; R- CTGGCGGAAGAAGAAGGTCAACA.

All 21 P. aeruginosa isolates that showed positive results for gyrB genes submitted to detect the presence of parC gene. PCR technique has been used for molecular detection using master mix (GoTaq-Green) with specific primers for the parC gene with product size of 357 bp:

F-CGAGCAGGCCTATCTGAACTAT; R-AGCAGCACCTCGGAATAG. All isolates were submitted to detect the presence of parE gene using master mix (GoTaq-Green) with specific primers for the parE gene with product size of 592bp:

F-CGGCGTTCGTCTCGGGCGTGGTGAAGGA; R-TCGAGGGCGTAGTAGATGTCCTTGCCG (17). All PCR reactions were done including 20 pmol for each primer, 12.5µl master mix, 3µl template DNA and complete the reaction with Nuclease free water up to the total volume 25µl for each reaction. PCR reaction tubes containing the mixture were placed in a thermo-cycler PCR instrument. DNA was amplified as the following conditions: Initial Denaturationat 95°Cfor 5 mins,30 cycles including (Denaturation at95°C for30 secs. Annealingat 58°Cfor 30 secs and Extension at72for 40 secs). In addition to one cycle of final Extension at72°C for7 mins. Five microliters of the product were subjected to electrophoresis, while the remaining product was stored at -20°C.The presence of mutations in parC gene were screened through submitting parC PCR amplicons for 9 isolates include FRDR regions to sequencing. These 9 isolates were selected as following: 6 isolates resistant to Ciprofloxacin including 4 isolates from burns sample (P2, P3, P4 and P14), 2 sputum samples (P15 and P21). In addition to three isolates sensitive to Ciprofloxacin isolated from burn, ear and urine sources. The presence of mutations was screened to determine the type of mutations in local P. aeruginosa isolates. parE PCR amplicons for 2 isolates (P1 and P16) from burn include FRDR regions were submitted to sequencing. PCR products were sent for Sanger sequencing, automated DNA sequence, by micro gen corporation –Korea. DNA sequences were analyzed and similarity searches were carried out with the Basic Local Alignment Search Tool (BLAST). The statistical data analysis was performed with statistical Analysis System- SAS program and Chi-square test.

Most of the isolates, 99 (82.5%), have belonged to Gram-negative bacteria, and the gram-positive isolates represent 16 (13.3%) while 5 (4.2%) of specimens showed no growth. Regarding Gram-negative bacteria, the burn specimens developed the highest isolation percentage, 80 (66.7%), while tissue specimens demonstrated the lowest isolation percentage, 2 (1.7%). According to the clinical and laboratory criteria, the distribution of P. aeruginosa among different types of specimens was analysed. The results showed that all of the 69 isolates were categorised into seven groups of different types of specimensas summarized in Table 1. The high prevalence percentage of P. aeruginosa in burn specimens 46 (66.7%).

Table 1

The distributions rates of *P. aeruginosa* isolates among the different specimen types.
Type of Specimens	No. of P. aeruginosa isolates (%)
Burn	46 (66.7)
Wound	4(5.8)
Mid-stream urine	4(5.8)
Sputum	4(5.8)
Endotracheal tube	5(7.2)
Ear swab	6(8.7)
Tissue	0 (0)
Total (%)	69 (100)
Chi-Square (χ²)	12.537 **
P-value	0.0001
** (P ≤ 0.01).

The results of the antibiotic susceptibility test of this study showed that 34 (73.9%), 36 (78.3%), 30 (65.2%) and 29 (63.04%) of P.aeruginosa isolates were resistant to Ciprofloxacin, Gentamicin, Imipenem, and Ceftazidime, respectively as showed in Table 2.

Table: 2 the percentage of sensitivity, intermediate resistant and resistance of P. aeruginosa according to categories of antipseudomonal antibiotics.

The concentration of extracted DNA samples of P. aeruginosawas between (50–80 ng/µl) and the purity of the DNA was (1.7-2). The 260:280 ratio is considered a good indicator of protein contamination (35).The extracted DNA of 21 P. aeruginosa isolates has been subjected to detect the gyrB gene. The gyrB gene was detected in all 21 (100%) of P. aeruginosa isolates. In a successful PCR reaction, the gyrB gene product of ~ 510 bp molecular weight was observed in gel electrophoresis (Fig. 1). This was considered a mandatory sign of a successful positive reaction. However, examined the negative control isolates (22C: klebsiella pneumoniae) showed negative PCR results which supports the accuracy and sensitivity of gyrB.

All 21 P. aeruginosa isolates that showed positive results for gyrB genes submitted to detect the presence of parC gene. The parC gene was detected in 21 (100%) of P. aeruginosa isolates with product of ~ 357 bp observed in gel electrophoresis as indicter of presence parC gene (Fig. 2).

Aligning of the obtained sequences nine with the reference strains (CP034435) in Gen Bank were analyzed for the presence of polymorphism of these genes and detection the mutations in the nucleotides. The results of the sequences alignment of the local isolates with the reference strain showed the presence of some mismatch in the nucleotides of our queries in the different positions including substitutions and deletions. In addition, it was found that the most of sequences had identical similarity (100%).

According to the results shown in Table 3, the isolates resistance towards Ciprofloxacin (P2, P3, P4 and P14) from burn and (P15 and P21) from sputum showed 100% identical to reference strains (CP034435). This could be mean absence of the different mutation in local isolates in comparison with reference P. aeruginosa strain. However, the sensitive isolates (P6, P11 and P13) towards Ciprofloxacin from burn, ear and urine respectively showed missense mutation (T/C: Leucine to Serine) in all of three isolates in addition to other missense mutation (A/G: Lysine to Serine) in isolate (P13) from urine.

Table 3

Mutations in the sequence of *parC* gene among local isolates in comparison with ATCC *P. aeruginosa* Strain (Accession number: CP034435).
Sample	Wild type	Mutation	Position	Mutation type	Effect of mutation
P2-Burn-R	Identical to the reference
P3-Burn-R	Identical to the reference
P4-Burn-R	Identical to the reference
P14-Burn-R	Identical to the reference
P15-Sputum-R	Identical to the reference
P21-Sputum-R	Identical to the reference
P6-Burn-S	T	C	513399	Transition	Missense Mutations Leucine < Serine
P11-Ear-S	T	C	513399	Transition	Missense Mutations Leucine < Serine
P13-Urine-S	T	C	513399	Transition	Missense Mutations Leucine < Serine
P13-Urine-S	A	G	513278	Transition	Missense Mutations Lysine < Serine
P: Pseudomonas aeruginosa R: Resistant S: Sensitive

All 21 P. aeruginosaisolates that showed positive results for gyrB genes submitted to detect the presence of parEgene. The parEgene was detected in 21 (100%) of P. aeruginosaisolates with product size ~ 592 bp in gel electrophoresis as indicter of presence parE gene (Fig. 3).

Aligning of the obtained sequences two with the reference strains (CP034435) in Gen Bank were analyzed for the presence of polymorphism of these genes and detection the mutations in the nucleotides. The results of the sequences alignment of the local isolates with the reference strain showed the presence of some mismatch in the nucleotides of our queries in the different positions including transition and transversion of the subject. According to the results shown in Table 4, the isolates resistance towards Ciprofloxacin (P1 and P16) from burn in comparison with reference strains (CP034435) showed differences. The resistant isolate (P1) showed 9 mutations including 6 silent transition mutation (3 Glutamine to Glutamine; 1 Glycine to Glycine; 1 Lysine to Lysine and 1 Asparagine to Asparagine). While 3 transversion mutations were determined, two of them were silent mutation (1 Glycine to Glycine and 1 Arginine to Arginine) in addition to one missense mutation (1 Valine to Serine). While P16 showed one (Adenine) deletion causing frameshift mutation.

Table 4

Mutations in the sequence of *parE* gene among local isolates in comparison with ATCC *P. aeruginosa* Strain (Accession number: CP034435).
Sample	Wild type	Mutation	Position	Mutation type	Effect of mutation
	G	A	510807	Transition	Silent Mutations Glutamine < Glutamine
	T	C	510832	Transition	Silent Mutations Asparagine < Asparagine
	G	C	510838	Transversion	Silent Mutations Glycine < Glycine
	T	G	510841	Transversion	Silent Mutations Arginine < Arginine
P1-Burn-R	A	G	510906	Transition	Silent Mutations Lysine < Lysine
	A	G	511006	Transition	Silent Mutations Glutamine < Glutamine
	A	G	511032	Transition	Silent Mutations Glutamine < Glutamine
	A	G	511058	Transition	Silent Mutations Glycine < Glycine
	A	T	511086	Transversion	Missense Mutations Valine < Serine
P16-Burn-R	A	-	5555278	Deletion	framshift
P: Pseudomonas aeruginosa R: resistance

The results of conventional diagnosis using selective media relatively agreed with many results of conducted studies (37, 5, 9, 1). Also, the present findings agreed with results found that most of the P. aeruginosa isolate produced specific pigment (pyocyanin) in Nutrient agar (36, 13). According to previous studies, the source of the isolates was not associated with the secretion of this pigment.

Numerous previous studies revealed that there is the β-hemolysis activity of P. aeruginosa isolates with a metallic sheen, blue-green colonies on blood agar, and appeared as small pale colonies on MacConkey agar due to lactose non-fermenting trait. While, the colonies on cetrimide agar appear mucoid, smooth in shape with flat edges and elevated centre, creamy colour and have a fruity odour (20, 23, 8, 7, 4).

This result agreed with the study of (21), who applied the microscopic examination to diagnose P. aeruginosa, which showed Gram-negative, small rods occur as single bacteria or in pairs. Also, this study agreed with (34), who showed that all the examined P. aeruginosa isolates appeared as Gram-negative after staining by Gram stain. The results showed that all examined isolates were positive for both oxidase and catalase.

The high prevalence percentage of P. aeruginosa in burn specimens 46 (66.7%) is supported by significant local studies. According to our results,the distributions rates of P. aeruginosaisolates among other specimen types were 4 (5.8%) for each of the following specimens: wound, urine and sputum. For urine samples, the findings match the results of a study conducted by (32) in India, who reported 4.6% for the prevalence rate of P. aeruginosa isolates. The ear and ETT specimens showed 6 (8.7%) and 5 (7.2%), respectively. While the results showed zero percentage from the tissue specimen.

In the current study, the resistance rate towards Aminoglycoside, including Gentamicin, was 36 (78.3%), which is considered the highest percentage compared to other categories. Also, the resistance rate towards fluoroquinolones, includingCiprofloxacin, showed a high percentage 34 (73.9%).In contrast, the results of antibiotics resistance showed 30 (65.2%) and 29 (63.04%) towards Carbapenems (Imipenem) and Cephalosporins (Ceftazidime), respectively.

The resistance rate against aminoglycosides (Gentamicin) (78.3%) to some extent were similar with local studies, which concluded that the highest resistance of P. aeruginosa examined isolates (burn samples) was against Gentamicin which reached 60% (37). However, this finding was higher than a local study (46.96%) conducted by (9). While this finding is slightly lower than the finding of the study achieved by (6), In addition, the result of resistance rate towards Ciprofloxacin, the primary generation of fluoroquinolones category, showed a high percentage of 34 (73.9%). This finding was slightly similar to the number of local and international studies (15, 31)reported the resistance rates Ciprofloxacin were 61.3%, 64% and 69.64%, respectively. On the contrary, current results were higher than previous local and international studies, which demonstrated the resistance rates of isolates to Ciprofloxacin ranged 28–35%(9, 14, 19).

P. aeruginosaisolates showed high resistance for most of the tested antibiotics. Bacteria can prevent the action of antibiotics through many mechanisms, including eliminating the accumulation of these antibiotics by efflux pump system(24).Fluoroquinolones (such as Ciprofloxacin) are currently among the most commonly prescribed antibiotics worldwide due to their broad spectrum of activity (19). The results revealed that 34 (74%) of P. aeruginosaisolates were commonly resistant to three of the tested antibiotics and above considered multidrug resistance (MDR). In comparison, 12 (26%) isolates were sensitive to more than one tested antibiotic. Statistical analysis showed highly significant differences (P-value equal to 0.0012).The result of MDR in the current study (74%) was higher than findings found in local and international studies, which reported the MDR prevalence ranged (30-52.5%)(11, 25).

P. aeruginosais capable of developing resistance to nearly all antibiotics categories through mutations in chromosomal genes in addition to plasmids (30). Furthermore, its ability to spread through hospital-acquired infections leads to considering it a significant health issue (26). In general, the antibiotic resistance profile variation among different isolates in Iraq is due to many factors, including diverse environmental conditions and health care among the region and others. Also, the physician and surgeon prescriptions described the misuse of antibiotics and wide-spectrum antibiotics.

gyrBgene is an essential gene in P. aeruginosaencoded for type II topoisomerase and plays an essential role in DNA replication. In the present study, the prevalence of this gene in target isolates is (100%). In current study, the specificity of gyrB gene P. aeruginosa in detection was examined through check subjected negative control, 22C (Klebsiella Pneumoniae) to PCR reaction and the result showed no amplified product as shown in Fig. 1. Usually, the 16S ribosomal RNA (16rRNA) gene is the most common target in bacterial genetic identification and characterization. However, recently gyrB gene acquired attention to use in detection due to its features such as it it is rarely transmitted horizontally and its molecular evolution rate is higher than that of 16S rRNA, and the gene is distributed ubiquitously(17, 27).

Although the PCR detection methods using genetic marker (e.g.: 16S rRNA, groE and opr genes) have been reported to be sensitive and specific, the gyrB gene has recently been noted to be a better candidate for the identification of bacterial species than the standard gene (16S rRNA)(27). The goal of most relative studies seeking rapid and precise detection method to detect P. aeruginosa to overcome the disadvantages of conventional protocols of bacterial identification. Thus, molecular techniques have essential role in species-specific detection of bacterial DNA and become a solution for detection issues.

Presence of parC in all isolates was agreed with study showed 100% percentage of the parC gene in 350 P. aeruginosa bacteria (14). Other study conducted by(29)showed that 115 isolates of fluoroquinolones resistance P. aeruginosa possess parC gene.

(13)reported that mutations in the parC gene play an essential role in resistance to Ciprofloxacin in clinical isolates of P. aeruginosa. There is study concocted by (12), PCR-sequencing was carried out to assess parC mutations in 30 drug-resistant isolates from different clinical sources. According to in silico analysis using MEGA7 software to the nucleotide sequences, mutations were observed in 79,16% parC (Ser87 → Leu), and 4,18% (Glu91 → Lys). Thus, they reported that antibiotic resistance to Ciprofloxacin and mutations in parC gene in resistant isolates are significantly related to each other (P < 0.05)(16). Many studies with clinical isolates have focused on ParC. They have demonstrated an amino acid replacement in ParC (Ser-87 to Leu), leading to a fluoroquinolone resistance in P. aeruginosa(3, 28).

The conclusion of the present study has come into the following: High prevalence of Pseudomonas aeruginosa in burns samples in comparison to other clinical samples. Increase the percentage of MDR Pseudomonas aeruginosa in particular resistance towards Fluroquinolones (Ciprofloxacin) could be due to misuse of these antibiotics. Furthermore, high capabilities of nosocomial Pseudomonas aeruginosa in acquiring resistance to various antibiotics lead to increase the pathogenies and become high risk on health. Therefore, applying molecular techniques in the identification of Pseudomonas aeruginosa using gyrB showed high accuracy and high sensitivity target gene. The resistance mechanisms of Pseudomonas aeruginosa to FQs are not related to the mutation in parC and parE only, but they are related to synergetic action with other type of mechanisms. Thus, mutations with targeted genes sequencing are reported in local isolates in comparison with reference strains. Increase the number of screened isolates is required for future study.

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Acknowledgments: A word of thanks goes to the staff unit of research in the college of health and medical techniques and Dr. Ali Dukhan’s laboratories for assisting me in my work.

Conflicts of Interest: The authors declare no conflict of interest.

The Local Ethics Committees approved the study with reference No. Csec/0221/0095.

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No competing interests reported.

Genotyping Investigation of Ciprofloxacin Resistance Genes (Parc And Pare) In Clinical Pseudomonas Aeruginosa Isolates

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Version 1

Abstract

Purpose

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials And Method

3. Results And Discussion

4. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1