Analysis of the Class 1 Integrons, Carbapenemase Genes and Biofilm Formation Genes Occurrence in Acinetobacter baumannii Clinical Isolates

Acinetobacter species are known as the primary causes of many severe infections and illnesses in recent years (Migliaccio et al. 2023). Acinetobacter baumannii has a high capacity for survival and causes outbreaks and long-lasting epidemics in hospitals. The difficulty with clinical treating these infections is increased by the growing number of strains resistant to many drugs because of their pan-drug and all-drug resistance (Lee et al. 2017). Human health is at risk because A. baumannii strains resistant to carbapenem are wide-spread (Harding et al. 2018). A. baumannii can build biofilms on non-living and biologic surfaces (Greene et al. 2016). Biofilm formation can promote the emergence of new and more antimicrobial-resistant phenotypes (Khoshnood et al. 2023). In a multicenter cohort study, all catheter-related urinary or bloodstream infections due to A. baumannii were caused by biofilm-forming strains, highlighting the unique role that bio-film formation plays in the development of infections related to medical devices (Rodríguez-Baño et al. 2008). A. baumannii cells in biofilms have the ability to go dormant and become metabolically inactive in unfavorable environmental conditions, allowing them to withstand environmental stress (Greene et al. 2016). Therefore, forming biofilms makes it easier for A. baumannii to adhere to medical implants and biological surfaces, facilitating colonization and infection. In addition, integrons also play a crucial role in the spread of bacterial drug resistance. Site-specific recombination allows integrons to express and capture external drug-resistance gene cassettes, a vital process in developing and disseminating drug resistance in bacteria. By identifying the presence of integrons and drug-resistance gene cassettes in multi-drug resistant strains, we can comprehend the associated drug-resistance mechanisms and their patterns of spread, as well as offer recommendations for clinical anti-infection treatments and nosocomial infection prevention (Gillings 2014). Understanding carbapenem-resistant A. baumannii (CRAB), including its epidemiology, distribution of integron, virulence factors, and carbapenem resistance mechanisms, is essential to preventing the continuing development of resistance to antibiotics in this species. Therefore, the primary purpose of this study was to identify the distribution of biofilm-formation-related virulence genes and integrons in CRAB isolates and their relationship with drug resistance.

CRAB is a significant cause of nosocomial infections that are associated with high mortality rates. It is commonly spread in ICUs (Chukamnerd et al. 2022). This is consistent with the report in this study. CRAB was mostly isolated from intensive care wards, while CSAB was widely distributed in different wards. The reason for this may be that most patients in intensive care wards have primary diseases, severe diseases with many complications, long hospitalization times, long-term indwelling catheters, and have been subjected to invasive operations for examination or treatment. In addition, the long-term use of broad-spectrum antibiotics increases the chance of infection. Sputum or throat swabs were the primary sources of the isolates, which is consistent with previous reports in our hospital (Xiao et al. 2019). It may be because A. baumannii is mainly related to respiratory infections, especially ventilator-associated pneumonia (Huang et al. 2019).

The mechanisms of drug resistance in A. baumannii include β-lactamases, aminoglycoside-modifying enzymes, permeability defects, the alteration and replacement of antibiotic target sites, enzymatic inactivation, and multidrug efflux pumps (Ibrahim et al. 2021). In this study, the resistance rates of 219 CRAB isolates to levofloxacin, cefepime, trimethoprim/sulfamethoxazole, amikacin, and minocycline were 79.91%, 77.17%, 75.80%, 59.82%, and 35.62%, respectively, while the resistance rates to other commonly used antibiotics were over 90%. Some isolates were resistant to tigecycline (5.02%) and colistin (1.37%). However, 50 CSAB isolates were sensitive to tigecycline and colistin, and the resistance rates to other commonly used antibiotics were only between 4% and 16%, indicating that the CRAB isolated in our hospital had severe drug resistance.

Bacteria can also spread drug-resistance genes between different isolates through horizontal gene transfer, spreading bacterial resistance. This gene transfer occurs through gene-moving elements such as plasmids or transposons. A large-scale survey in Colombia (Villegas et al. 2007) found that 33.6% of A. baumannii was resistant to carbapenems, and 98.5% of resistant isolates carried the bla_OXA-23 group. IMP and VIM enzymes have specific detection rates in the Far East and Europe and are related to the carbapenem resistance of A. baumannii (El-Ageery and Al-Hazmi 2014; Ibrahim 2019; Nikibakhsh et al. 2021). In 2013, 13 (38.2%) of 34 CRAB isolates from a hospital in Huaibei (a city in Anhui province, China) produced the KPC-2 carbapenem enzyme (Zhang et al. 2015). The detection rate for bla_NDM-1 in CRAB from Algeria was 10.6% (Khorsi et al. 2015). It is worth noting that bla_IMP, bla_VIM, bla_KPC, and bla_NDM were not detected in this study. Most of the 219 CRAB isolates in this study carried bla_OXA-23-like (99.54%), which is consistent with previous reports, and carbapenem-resistant A. baumannii in our country mainly carry bla_OXA-23-like (Xiao et al. 2019).

In this study, whole-genome sequencing analysis of a bla_OXA-23-like-negative isolate indicated that this isolate carried blaOXA-72, blaOXA-259, and blaADC-26 at the same time, which could confer β-lactam resistance. bla_OXA-72 is one of the most important allelic variants of bla_{OXA-24/40-like} (Lasarte-Monterrubio et al. 2022). However, bla_OXA-259 is a member of the bla_OXA-51 family and has only been reported in one hospital in Hangzhou, China (Jia et al. 2019). Acinetobacter-derived cephalosporinases (ADC) are cephalosporinases produced by Acinetobacter; bla_ADC-26 is a variant of ADC and was first discovered in France (Rodríguez-Martínez et al. 2010). In this study, the resistance phenotype of AB-134 provided resistance not only to carbapenems but also to ceftazidime, and the detection of bla_ADC-26 indicated that this gene might mediate ceftazidime resistance. In addition, MLST typing of this isolate resulted in the discovery of new STs, ST3272 (Oxford) and ST2526 (Pasteur).

One of the most important virulence factors of A. baumannii is its formation of biofilms, which facilitate the bacterial adherence to medical equipment and colonization of the surface of human skin for long-term survival (Dolma et al. 2022). Studies have confirmed that abal, cusE, bap, and bfmS genes are all related to the formation of biofilms. CsuE is a key gene for the fimbriae synthesis in A. baumannii (Pakharukova et al. 2018). The expression of fimbrial synthesis genes is regulated by the bfmRS two-component regulatory system (Roy et al. 2022). In addition, the quorum sensing system also governs the formation of bacterial biofilms, and the quorum sensing signaling-molecule-encoding gene abal plays a role in forming bacterial biofilms on abiotic surfaces (Sun et al. 2021). Studies have shown that mutations of the bap gene mainly affect the volume and thickness of the biofilms formed by A. baumannii (Azizi et al. 2016). In this study, the detection rates of the virulence genes abal, bap, and cusE in CRAB were higher than those of CSAB, with a statistical difference (p < 0.05). In CRAB, the resistance rates of cusE-positive isolates to levofloxacin and cefepime were higher than those of cusE-negative CRAB isolates (p < 0.05). The resistance rates of bap-positive isolates to tobramycin were higher than those of bap-negative isolates (p < 0.05). When bacteria exist in the form of a biofilm, drug resistance is significantly enhanced (10–1,000 times); antibiotics cannot effectively remove the bacterial biofilm and can also induce drug resistance (Aliramezani et al. 2016), thus aggravating clinical infection and increasing the difficulty of treatment. While the resistance rates of bfmS-negative isolates to trimethoprim/sulfamethoxazole were higher than those of bfmS-positive isolates (p < 0.05), this may have been due to bfmS negatively regulating antimicrobial resistance in A. baumannii (Kim et al. 2019).

Integrons can capture and express resistance gene cassettes through site-specific recombination and play important roles in the production and spread of drug resistance genes (Lee et al. 2017). In this study, we screened for intI1 in 269 A. baumannii isolates. Among them, 75 isolates of CRAB and three isolates of CSAB were positive for intI1, consistent with our previous reports (Xiao et al. 2019). The amplified 75 variable regions in 78 intI1-positive isolates were all aacA4-catB8-aadA1, which can confer resistance to chloramphenicol and aminoglycoside. It can explain, in part, how the drug resistance rates of the intI1-positive isolates to tobramycin and amikacin were higher than those of the intI1-negative isolates (p < 0.05). Apart from tobramycin and amikacin, the resistance rates of intI1-positive CRAB isolates to trimethoprim/sulfamethoxazole and minocycline were also higher than those of intI1-negative CRAB. The sul1-qacEΔ1 regions in the 3’ conserved segment of the class 1 integron may partly explain the higher resistance rates of intI1-positive CRAB isolates to trimethoprim/sulfamethoxazole. In addition, the class 1 integron was often embedded in transposons or located in drug-resistance plasmids, in which many other drug-resistance genes were also carried. This can partly explain the relatively higher resistance rates of intI1-positive isolates to other antibiotics that cannot be conferred by antibiotic-resistance gene cassettes in variable regions of class 1 integrons.

In this study, the Pc promoters of the variable regions in 78 intI1-positive isolates, such as PcH2, were all relatively strong, and the expression levels of downstream gene cassettes were relatively high, which can confer a higher level of drug resistance to bacteria (Wei et al. 2011; Barraud and Ploy 2015). Meanwhile, the efficiency of exogenous gene cassette integration into the attI1 site downstream of strong Pc promoters was also lower (Wei et al. 2011). The integrase protein corresponding to a stronger Pc (PcS or PcH2) has a weaker ability to capture exogenous gene cassettes to maintain the relative stability of integrons (Barraud and Ploy 2015). This may be one of the reasons for the identity of gene cassette array aacA4-catB8-aadA1 found in this study.

Because the Pc of class 1 integrons is opposite to the promoter of integrase, there is mutual interference, and studies have shown that the stronger Pc of class 1 integrons can affect the expression of integrase (Lacotte et al. 2017). The SOS stress response system regulates the expression of intI1, and binding the LexA protein to the adjacent sites of the −10 region of the intI1 promoter P_int can inhibit the transcription of intI1. Studies have shown that A. baumannii lacks LexA-driven integrase repression but restricts the expression of intI1 through the selective retention of the strong Pc to prevent the toxic effects caused by the massive expression of integrase protein (Couvé-Deacon et al. 2019). This may explain why the Pc of the class 1 integron in this study’s clinical isolates of A. baumannii was relatively strong.

In summary, bla_OXA-23-like may be the main reason for CRAB’s resistance to carbapenem. A new (Oxford 3272, Pasteur 2520) CRAB isolate ST carrying the bla_OXA-72, bla_OXA-259, and bla_ADC-26 was discovered.