Multiple Mechanisms Synergistically Induce Pseudomonas Aeruginosa Multiple Drug Resistance

Background: This study was designed to detect the molecular epidemiological characteristics and resistant mechanism of carbapenem resistant Pseudomonas aeruginosa (CRPA) which provide reference for the prevention and treatment of hospital CRPA infection. Methods: 34 strains of CRPA from 2018 to 2019 were isolated and their resistance to 13 commonly used antibiotics was detected using TDR-300B Plus VitEK-2 compact automatic bacterial identication instrument. Then carbapenemase production was detected using Carbe NP test. The eux pumps MexA and outer membrane protein OprD proteins were detected using RT-PCR and class (cid:0) integron carried with drug-resistant genes were detected using PCR and sequences analysis. Results: Among 34 strains of CRPA, 22 strains were multiple drug resistance (MDR) and 5 strains were extensively drug-resistant (XDR). The results of class (cid:0) integron carried drug-resistant gene sequencing analysis showed the class (cid:0) integron mainly carried aminoglycoside or quinolone antibacterial drug resistant genes. Conclusion: Multiple mechanisms play an important role in the formation and development of MDR or XDR resistance.

Background P. aeruginosa is the common opportunistic pathogenic bacteria in hospitals, which often get adhered to the surface of medical machinery equipment. Once the immunity of the human host is compromised, it can lead to number of infections including, respiratory tract infection, skin infection, urinary tract infection and burn infection. P. aeruginosa can also be spread in blood, inducing disseminated bacteremia, sepsis and even death [1]. In the recent years, a clinical concern aroused due to P. aeruginosa resistant to multiple drug resistance (MDR) and extensively drug-resistant (XDR), causing di culties to the clinical treatment [2]. Carbapenem are commonly used drugs in clinical treatment of MDR-P. aeruginosa (MDR-PA) and XDR-P. aeruginosa (XDR-PA). Although new-lactam antibacterial drugs with broad antibacterial spectrum, strong antibacterial activity and inhibiting almost all gram-negative bacteria constantly are developed, P. aeruginosa often develops resistance against carbapenems [3].This results in the decrease of treatment e cacy and the increase of the dosage [4]. Based on the emerging studies, the drug resistances to carbapenems of P. aeruginosa are usually associated with carbapenemase production, excessive expression of active e ux system; outer membrane protein expression and integron carried drug-resistant genes. Besides, bacterial bio lm to a great extent, prevents antimicrobial drugs from entering [5,6]. Therefore, we investigated the drug resistance in P. aeruginosa isolated from the clinic, detected multiple mechanisms and analyzed the possible mechanisms of MDR and XDR.

Bacterial strains
A total of 34 CRPA clinical bacterial isolates were collected from various clinical laboratories in Hunan  were de ned as "CRPA," and those resistant to three or more drugs class were de ned as "MDR-PA or XDM-PA" [7]. P. aeruginosa ATCC 27853 was used as the control for antibiotic resistance.

Detection of carbapenemase production
Carbapenemase production was detected using Carbe NP test as described by Bouslah [8]. That carbapenemase in the bacteria was completely released through the non-denouement tissue lysate and hydrolyzed imipenem to produce acid. This changed the pH and led to the phenolic red color change from red to yellow or orange, indicating carbapenemase as positive.

Quanti cation of Mex A and OprD
Total RNA was extracted from exponential growth of bacteria in Luria Bertani medium using TRT-101(TOYOBO, China) and residual DNA was removed by DNase I. Then a cDNA synthesis was using reverse transcription kit (TOYOBO, China) with some modi cations. PCR reaction system was as follows: the total volume of was 25 µl, including 1µl of reverse transcription product, 0.25 µl of upstream and downstream primers, 2× mix PCR buffer 12.5 µl, and 11.25 µl ddH 2 O. The reaction conditions were predenaturation at 94℃ for 2 min, 35 cycles of denaturation at 94℃ for 30 s, annealing at 54℃ for 30s, extension at 72℃ for 30s and at last extension at 72℃ for 10 min.
The cDNAs were subjected to semi-quantitative PCR using primers (Table 1), relative gene expressions were evaluated using RpsL representing housekeeping gene. P. aeruginosa-PAO1 was used as a reference for normalization of relative mRNA levels. The MexA were considered over expressed when their transcriptional levels were at least diploid higher than those of PAO1, and the expression of OprD decreased when their transcriptional levels were equal to or less than 30% those of PAO1 [9]. 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and last extension at 72 ℃ for 10 mins. The products were sequenced at Sunnybio (China).The nucleotide sequences of variable regionwere analyzed with BLAST tool of NCBI (https://www.ncbi.nlm.nih.gov/) by comparison with sequences of the reference strain and PAO1 retrieved from the data bank.

Statistical analysis
All experimental data were analyzed by WHONET 5.6 and SPSS 22.0 software.

Antibiotic sensitivity
The drug resistance rates of the 34 P. aeruginosa strains to meropenem and imipenem were 100% and 85.29% respectively. Antibiotic resistances were as follows, there were 22 strains with antibiotics resistant to 3 and more, de ned as multi-drug resistant bacteria (MDPA). Five strains of bacteria were resistant to 6 types and more of antibiotics, de ned as extensively resistant strains (XDPA).

Gene expression analysis
The relative expression levels of MexA and OprD genes were determined by semi-quantitative ( Table 3). The results revealed that 23.53% (n = 8) of isolates displayed increased MexA mRNA, and 47.06% (n = 16) of isolates displayed decreased transcription of OprD mRNA. According to the drug sensitivity test, 34 CRPA strains were divided into three groups: MDPA, XDPA and CRPA (Figure 1a-c). Compared with the control group PAO1, OprD expression was signi cantly down-regulated, although in CRPA, XDPA or MDPA, there were varying degrees of down-regulation. The expression of MexA was slightly up-regulated in CRPA, but a signi cant up-regulation in the XDPA and MDPA groups, which may help increase the resistance of P.aeruginosa.
PCR ampli cation and sequencing of the class I integron Class 1 integrons were detected in 13 isolates (Fig.2). The positive strains were sequenced, and the sequence comparison results showed that they carried 3 kinds of drug-resistant gene cassettes ( Table 2). Acc(6')-lb, catB3, aadB and clmA6 cause P.aeruginosa to be insensitive to aminoglycoside drugs, while qnrvc1 leads to resistance to quinolones. See Supplementary Figure 1 for sequencing results. All strains with down-regulated OprD had high imipenem MIC (≥ 32 µg/ml) resistance. All the MexA over expressed strains showed high resistance to imipenem (≥ 32µg/ml) and meropenem (≥ 32µg/ml). The bacterial strains with multiple drug resistance mechanisms at the same time were very low, among which, it carried class I integron, up-regulated MexA and down-regulated OprD was 2.94% (n = 1), manifested as XDR. The strains with class I integron and increased MexA were 5.88% (n = 2), manifested as MDR or XDR. The proportion of class I integron and down-regulated OprD were 5.88% (n = 2), which was manifested as MDR. The strains with down-regulated OprD and over expressed e ux pump MexA were 2.94% (n = 1), presenting as MDR. This suggests that multiple mechanisms play an important role in the development of MDR or XDR (Table 3).

Discussion
Invasive operation in hospital is an important cause of opportunistic infection of P. aeruginosa. Generally, the patients who receive invasive operation need to use antibiotics to control infection. However, such patients are usually in poor health and compromised immunity. Especially in elderly patients, P. aeruginosa has become the main pathogen of infection in middle-aged and aging patients, accounting for the rst place of pathogens [11].
Different kinds of antibacterial drugs are often used alone or in combination for the treatment of patients with P. aeruginosa infection, and β -lactamase antibiotics are the most common, especially carbapenem. In this study, 34 strains of CRPA in hospital were taken as the research objects, and their drug resistances were analyzed. The results showed that the drug resistance rates of CRPA to meropenem and imipenem were 100% (n = 34) and 85.29% (n = 29), respectively, indicating that carbapenem antibiotics should be used more carefully in clinic. The multi-drug resistance rate was 52.94% (n = 18), and the pan-drug resistance rate was 14.71% (n = 5). As it can be seen from this situation, the drug resistance of P. aeruginosa has been severe, and more attention should be paid to the detection of P. aeruginosa resistance in clinical treatment, rational use of antibacterial drugs, and reduction of the spread of multiple drug-resistant P. aeruginosa in hospitals.
The mechanism of P. aeruginosa resistance to carbapenems is complex including metal enzyme (MBLs), active outer membrane e ux system, and decreased outer membrane permeability [12][13][14]. In addition, P. aeruginosa can also capture external drug-resistant genes through gene horizontal transfer elements integrons. The common integrons in P. aeruginosa is class integron, which has many kinds of drugresistant gene boxes and a wide range of hosts, making bacteria multi-drug resistance [15][16][17]. In this study, class I integron detection rate was 38.24% (n = 13) among 34 strains, mediated to quinolone and aminoglycoside resistance. Moreover, class I integron genes are more integrated on the bacterial chromosome, making the genetic stability and maintaining the resistance[18]. However, we did not detect carbapenemase in the integron I. The possible reasons are that the carbapenemase exists in other types of integrons, exists in the genome of living bacteria, or there are other mechanisms of drug resistance to β -lactamase antibiotics in bacteria, which will be further explored in the following research.

Conclusion
In 34 strains, the ratio of strains with multiple drug resistance mechanisms was very low. The bacterial strain with class I integron, up-regulated MexA and down-regulated oprD was 2.94% (1/34). Strains with two of the three mechanisms at the same time were 14.7% (5/34). The above results suggest that multiple mechanisms play an important role in the formation and development of CRPA. Moreover, the drug resistance genes carried by class I integrons and the synergistic effect of multiple mechanisms play a synergistic role in the formation of MDR and XDR. We will further explore the formation mechanism of multi-drug resistance of P. aeruginosa by inducing the synergy of multiple mechanisms in vitro.

Declarations
Ethics approval and consent to participate: This article does not contain any studies with human participants or animals performed by any of the authors.

Consent for publication: Not applicable
Availability of data and material: the data are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no competing interests