Investigation of antimicrobial susceptibility, class I, II, and III integrons among clinical isolates of Pseudomonas aeruginosa from hospitalized burn patients in Southern Iran

Pseudomonas aeruginosa is a non-fermenting opportunistic gram-negative bacillus known as an important nosocomial pathogen that can cause many chronic and acute infections in patients with severe burns, neutropenia, cystic fibrosis, and compromised immunity [1, 2]. People with serious burns will be exposed to P. aeruginosa since it is so prevalent in the environment before their wounds get better [3]. Burn infection due to P. aeruginosa poses a considerable challenge in terms of graft loss, systemic sepsis, prolonged hospital stay, and even enhanced mortality [4]. Burn hospitals usually harbor multidrug-resistant (MDR) P. aeruginosa that can be a cause of infection. P. aeruginosa has been found to contaminate floors, sinks of hospitals, and the bed rails. Additionally, this bacterium has been cultured from nurses’ hands [5, 6].

Organisms can resist the effects of antimicrobial drugs in three ways: limited absorption and efflux; drug inactivation; and changes in targets [7]. A particular bacterium strain’s vertical transmission of genetic variants between generations and mutational events produced during bacterial replication may be to blame for the emergence of resistance in bacteria. Plasmids, integrons, and transposons are examples of accessory genetic elements that can move horizontally in bacteria and transmit antibiotic resistance determinants [8]. This can result in widespread resistance. Microorganisms can now change more quickly than is conceivable through mutation alone thanks to the evolution of transposons and integrons. Although integrons and transposons are usually carried on plasmids, they can also be found on the chromosome. The overuse and uncontrolled use of antimicrobial agents is probably the principal reason for the development of MDR P. aeruginosa eruptions in clinical locations [9,10,11]. The spread of antibiotic resistance by clinical pathogens has been linked to the mobility of integrons, which is related to mobile DNA elements (transposon and plasmid). Integrons (mainly Class I) are known as mobile genetic elements that are most frequently seen on transferable plasmids, despite the fact that they lack the ability to move [12]. Integrons can capture, integrate, and mobilize gene cassettes of drug resistance and combine them using site-specific recombination [13]. As a result, they rapidly spread antibiotic resistance among different bacterial strains, especially in the gram-negative [12]. The construction of integrons contains a mutable region of gene cassettes and 3′ and 5′ conserved segments between the two conserved segments. The common promoter (Pc), the recombination site (attI), and the integrase gene (IntI) are encoded within the 5′ conserved segment, whereas the quaternary ammonium compound resistance sequence (qacEΔ1) and sulphonamide resistance gene (sulI) are encoded within the 3′ conserved segment [14,15,16]. Gram-negative bacteria have been shown to contain integrons that may be classified into 3 groups based on the intI genes’ amino acid sequence [17]. Among classes of integron, classes I and II are the most ubiquitous in the MDR gram-negative bacteria associated with failure of antibiotic treatment [18]. Class I integrons carry more than 40 resistance genes associated with resistance to beta-lactams, aminoglycosides, macrolides, chloramphenicol, sulfonamides, and disinfectants [19], whereas gene cassettes in class II integrons are mainly associated with various resistances such as streptomycin, spectinomycin, and trimethoprim [20]. Class III integrons are rarely found in clinical specimens but recently have been found in clinical specimens such as P. aeruginosa, P. putida, Klebsiella pneumoniae, and Serratia marcescens [21]. Due to the important roles of integrons in antibiotic resistance among gram-negative bacteria and enhanced resistance to drugs, the aim of the present study was to consider the frequency of class I, II, and III integrons in P. aeruginosa isolated from hospitalized burn patients obtained from a teaching hospital in Ahvaz, Iran.

In our study, 70 P. aeruginosa strains were isolated from biopsies (63%), blood (27%), wounds (6%), and sputum (4%) samples. P. aeruginosa isolates had been collected from burn patients in different wards of the hospital, as follows: ICU (51.4%), burn ward (40%), and surgery (8.6%). In addition, among the 70 P. aeruginosa isolates, 34 (48.6%) were obtained from females and 36 (51.4%) from males.

PCR results of the three classes of integron genes showed that 51.4% of isolates (36/70) had intI1, 30% of isolates (21/70) had intI2, and 12.9% of isolates (9/70) had both genes. Also, the intI3 gene was not detected in any isolates. The patterns of antibiotic resistance of intI-positive and -negative P. aeruginosa isolates are shown in Tables 2 and 3. Antibiotic susceptibility patterns among intI1-positive isolates showed a high level of antibiotic resistance to gentamicin and imipenem (97.2%), while the lowest antibiotic resistance was to ceftazidime (80.6%), followed by ciprofloxacin (83.3%). Among intI1-negative isolates, the highest frequency of antibiotic resistance was to gentamicin (97%), followed by ceftazidime (94.1%), whereas, the lowest rates of antibiotic resistance were observed to ciprofloxacin (79.4%). In intI2-positive isolates, the highest resistance was to gentamicin and ceftazidime (95.2%). The lowest rates of antibiotic resistance in this group were to ciprofloxacin (71.4%). In intI2-negative isolates, the highest antibiotic resistance was seen against gentamicin and imipenem (95.9%), while the highest antibiotic sensitivity was to ceftazidime (83.7%) followed by ciprofloxacin (85.7%). In this study, we found no significant association between the presence of intI genes and antibiotic resistance.

P. aeruginosa is one of the most important pathogens in patients with burn infection. An important problem in controlling these infections in the burn units is the emergence of MDR isolates of P. aeruginosa [24]. Different antimicrobial resistance genes can be located on gene cassettes and transported by integrons placed on transposons, chromosomes, and transmissible plasmids [25]. In this study, 51.4% and 30% of P. aeruginosa isolates carried class I and II integrons, whereas 12.9% of isolates contained both classes of integron genes. Examining studies conducted in Iran and other countries, it was found that the prevalence of the intI1 gene is ranging from 27.5% to 62%, which is close to our results [26,27,28,29,30,31]. Increasing the frequency of class I integrons can cause an increase in antibiotics resistance and its spread among bacterial strains. The incidence of intI2 genes in P. aeruginosa isolates varies due to incorrect antibiotic use, geographic dispersion, and the origin of infections. For example, in contrast to our findings, Mobaraki et al. and Hosseini Pour et al. showed 25.5% and 52% isolates carried class II integrons, respectively [29, 32]. Conversely, Zarei-Yazdeli et al. showed class II integrons were found in 22 (15.3%) P. aeruginosa isolates, which is very close to our results [33]. In astudy performed by Khosravi et al., the prevalence of class I and II integrons was reported at 95% and 0%, respectively, while in our study, the intI1 and intI2 genes were identified in 40% and 15% of isolates, respectively [34]. Comparing these two studies, it can be concluded that the occurrence of integrons is shifting from class I to class II and so the prevalence of integron class II is increasing among hospitalized burn patients. In our study, no intI3 gene was detected in any of the isolates. The antimicrobial susceptibility pattern among intI-positive isolates showed that resistance to gentamicin was high. Khosravi et al. also showed in their study that the highest resistance to antibiotics among intI-positive isolates was to gentamicin [34]. In a study performed by Zarei Yazdeli et al., the most effective antibiotics were tobramycin and ciprofloxacin, and the resistance to gentamicin was 79.9%, which was lower than our study [33]. This difference may be due to the type of patients and the difference in the geographical area. Among intI-negative isolates, the highest resistance was related to gentamicin. Similar to our results, Zarei Yazdeli et al. reported that the highest antibiotic resistance among intI-negative isolates was to gentamicin [33]. However, in the study performed by Khosravi et al., among intI-negative isolates, no resistance to antibiotics was reported, which may be due to the small number of intI-negative isolates (4 isolates) [34]. The most successful antibiotic in the current investigation was colistin, which had a 100% susceptibility rate in both intI-positive and -negative isolates, whereas the majority of P. aeruginosa isolates displayed a significant rate of resistance to other antibiotics. According to our findings, colistin was the most productive antibiotic for the treatment and management of nosocomial infections, according to Goli et al., Mobaraki et al., and Khosravi et al. [26, 29, 34]. In this study, we found no substantial association between the presence of intI genes and antibiotic resistance. This may be for two reasons: 1) Integrons without a gene cassette, called “empty integrons”, have no resistance cassettes [35]; 2) Gene cassettes that cause antibiotic resistance may be carried by transposons or prophages instead of integrons [36]. Contrary to our results, Zarei-Yazdeli et al. reported a substantial association between the presence of the intI1 gene and resistance to gentamicin, and also between the intI2 gene and resistance to ceftazidime, tobramycin, and amikacin [33]. Also, in the study of Faghri et al., a substantial association was observed between the presence of class I integron genes and resistance against meropenem [1]. In another study performed by Yari et al., there was a significant association between the presence of class I integrons and resistance to amikacin, imipenem, gentamicin, ceftazidime, and ciprofloxacin [37]. Integrons are thus one of the most significant contributors to antibiotic resistance. Due to the arbitrary and inappropriate use of antibiotics in the last decade, we have faced many problems and concerns in the treatment and control of infections caused by strains with high levels of antibiotic resistance. Therefore, recognizing the causes of antibiotic resistance, such as integrons, and examining their frequency can be helpful.

Generally, this study’s findings show important data on the presence of class I integrons in P. aeruginosa isolates obtained from hospitalized patients. The role of integrons as mobile genetic components in the horizontal transfer of antibiotic resistance has received attention in recent years, especially resistance to beta-lactam and aminoglycosides According to descriptive results, the isolates carrying class I integrons had higher antibiotic resistance rates than class II integrons for piperacillin/tazobactam (94.4%), imipenem (97.2%), ciprofloxacin (83.3%), and gentamicin (97.2%) and there were no statistically significant differences. Because intI have more than one resistance gene cassette and are often transported by motile genetic elements, they lead to the spread of resistance factors from one side to the other. In order to establish infection control programs and stop the development of resistant strains, it is crucial to identify these types of antibiotic resistance genes. The development of multi-resistance by Enterobacteriaceae is predominantly driven by horizontal transfer of integron-carrying elements, regardless of the species or origin of the bacteria.