Relationship between Biofilm Regulating Operons and Various b-Lactamase Enzymes: Analysis of the Clinical Features of Infections caused by Non-Fermentative Gram-Negative Bacilli (NFGNB) from Iran

Bacteria are capable of evolving high doses of the drug in various infections by forming biofilms. Perhaps, biofilm regulator genes have different frequencies in β-lactam producing non-fermentative Gram-negative Bacilli (NFGNB). In this study, we investigated the role of biofilm operons of Pseudomonas aeruginosa and Acinetobacter baumannii on the prevalence of different β-lactamase enzymes. Onehundred twenty (120) nosocomial NFGNB isolates were collected from different clinical samples of patients. PCR method was used for the amplification of resistance genes. Isolates were collected, including 50 isolates (41.66%) of P. aeruginosa and 70 isolates (58.33%) of A. baumannii. The distribution of ESBL, AmpC, KPC, and MBL β-lactamase enzymes in P. aeruginosa and A. baumannii isolates were 64%, 58%, 38%,44%, and 57.14%, 60%, 32.85%, 34.28%, respectively. The frequency of csuABC, pgaABC operon in A. baumannii were as follows: pgaA (45.71%), pgaB (32.85%), pgaC (42.85%), csuA (34.28%), csuB (32.85%), csuC (41.42%), and ompA (38.57%). Further, the prevalence of pslABC and pelABC operons in P. aeruginosa isolates were as follows: pslA (58%), pslB (58%), pslD (60%), pelA (64%), pelB (38%), pelC (44%), and algD (68%). This study revealed that the abundance of biofilm regulator genes in NFGNB strains is affected by different β-lactamase enzymes.

set (UK, MAST, code: D68C) based on manufacturer instruction. Klebsiella pneumoniae ATCC 70063, Escherichia coli ATCC 25922, and Enterobacter Creole NCBT 13406 were used as a positive control. Screening and confirmation of MBL and KPC producer strains For the detection of MBL producing strains, EDTA-imipenem microbiological (EIM) was used. For the detection of carbapenemaseproducing strains, the Modified Hodge test (MHT) was used 13 .

Screening of biofilm producer strains
Biofilm production was assessed using a crystal violet microtiter plate assay according to the method of Ghadaksaz et al study 11 . The OD of each well was measured at 550nm and 595nm using the microplate reader (Omega Fluostar, Germany). Bacterial biofilms were classified based on an OD cut-off ODc as described. In this case, P. aeruginosa PAO1 and A. baumannii ATCC 19606 was used as the positive control, and the culture medium used as negative controls.

DNA Extraction
DNA was prepared for PCR according to the method described previously with some modifications 13 . Briefly, the organisms were grown overnight, and from that young culture of P. aeruginosa were taken in a 2 ml microcentrifuge tube and centrifuged it at 3000 rpm for 10 minutes. Then the supernatant was discarded, and 100µl of 1X Tris-EDTA buffer (10 mM Tris-HCl, 1mMEDTA [pH 8]) was added to the pellet. The microcentrifuge tubes were placed in a water bath at 100°C for 10 minutes and immediately cooled on Ice. Following centrifugation, the supernatant was used as a template for PCR.

Biofilm operons Genes Detection
Primer sets used were obtained from Macrogen, Korea (Table 1). Template DNA in a volume of 2μL was added to the 12.5μL master mix (Ready Mix TMTaq PCR Reaction Mix, Sigma) with 0.4μM of each primer for a final volume of 25μL in each PCR. DNA templates were subjected to one regime of amplification. After initial denaturation at 95°C for 5min. The PCR cycling consisted of initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 1min annealing at 55 to 61°C (according to Table 1), 72°C for 90 sec and a final extension at 72°C for 5 min. The PCR products (10μL) were analyzed by electrophoresis on 1%agarose gel. Finally, the amplified bands in the gel were visualized by a trans-illuminator (UV light) to confirm the PCR products.

Statistical analysis
Based on Fig. 3A and 3B, there was a significant relationship between the biofilm formation and β-lactamase enzymes in P. aeruginosa and A. baumannii (p≤0.001). Besides, there was a significant association between biofilms regulatory genes and β-lactamase enzymes (p≤0.05). No statistical association was detected when the virulence factors were compared to some antibiotic. Moreover, we found no significant difference in antibiotic susceptibility between the fluoroquinolones, aminoglycosides (except for amikacin), and biofilm regulatory genes, a very similar distribution of disinfectant resistance genes than others (p≤0.05).
Besides, a high abundance of biofilm regulator genes was observed in strains resistant to carbapenems, monobactams, and amikacin ( Table 2 and Table 4). However, in some isolates, harboring carbapenemase enzymes and biofilm operon genes was negatively associated with biofilm formation (p<0.05). In some MBL and AmpC producer isolates, harboring β-lactamase enzymes was negatively associated with biofilm production (p<0.05).

DisCUssiON
Alterations of chromosomal genes are still by far the most critical mechanisms of β-lactam resistance in NFGNBs, although transferable carbapenem resistance is becoming increasingly important 1 .
As shown in Fig. 1, this paper reported that high resistance rate to ciprofloxacin (72% and 87.4%), gentamycin (66% and 77.1%), and trimethoprim/sulfadiazine (40% and 55.7%) in Iranian isolates of P. aeruginosa and A. baumannii, respectively. Some researcher demonstrated that ciprofloxacin and trimethoprim/sulfadiazine are the two most frequently co-transferred resistance phenotypes among P. aeruginosa and A. baumannii isolates 14 . The significantly high level of resistance to these antimicrobials was probably an indication of their extensive usage in the clinic for therapeutic and prophylactic purposes both for NFGNBs other infections.
Moreover, based on Table 1, MDR and XDR strains were detected in 22%, and 14% of P. aeruginosa and 31.4% and 15.7% of A. baumannii isolates, respectively. A similar pattern of results were obtained in many studies, who reported a high frequency of MDR and XDR strains in NFGNBs 15,16 . In contrast to our findings, some studies in Egypt 17 indicate the different prevalence of MDR and XDR strains in NFGNBs. In Table 2, we reported that the frequency of AmpC-producing, ESBL-producing, KPC-producing, and MBLproducing P. aeruginosa isolates were 56%, 64%, 48%, and 38%, respectively; which was in line with Tohamy et al. 17 .
Furthermore, AmpC-producing, ESBLproducing, KPC-producing, and MBL-producing A. baumannii isolates were 57.1%, 60%, 34.2%, and 32.8%, respectively. This result ties well with Goel et al. 18 study and shows that the distribution of β-lactamase enzymes in A. baumannii is higher than P. aeruginosa. However, the rates of ESBL and AmpC producer strains were higher when compared to the rates reported from India 19 and Lebanon 20 . Our study found that more than 50% of the strains from the west of Iran were β-lactamresistant is undoubtedly a cause for concern as β-lactam has been the drug of choice for A. baumannii and P. aeruginosa infections for over a decade.
Biofilm formation and regulatory genes have been investigated as controversial and critical issues in healthcare settings. However, in some studies, no apparent relationship between β-lactamase enzymes and biofilm formation has been detected 21,22 . Further, as shown in Fig. 3, the current study confirmed the strong association between biofilm formation and β-lactamase enzymes (p< 0.001), which was reported similar results in several studies 23,24 . Also, based on Table  2 and Table 4, our finding showed that resistance to meropenem, ceftazidime, and amikacin, was associated with a higher prevalence of the biofilm formation and regulatory genes. Nonetheless, many studies showed that antibiotic resistance was associated with the production of the biofilm phenotype, such as cellular appendages and adhesions 9 .
In this study, a high prevalence of pgaA, pgaC, and csuC genes (45.7%, 42.8%, and 41.4%, respectively; p< 0.05) in resistant A. baumannii was observed. These results go beyond Liu et al. 25 reports how demonstrated the frequency of pathogenic genes (ompA) and biofilm regulators in the β-lactamase producer strains of A. baumannii was higher. Additionally, in Table 3 and Table 4, we confirmed that algD, pelA, and pslD (68%, 66%, and 60%, respectively; p< 0.05), which was also more prevalent in resistant strains. Another studies shown a significant relationship between virulence factors and antibiotic resistance in P. aeruginosa 13,26 . This evidence highlights that the possibility of acquisition of both resistance and virulence traits via horizontal gene transfer could be responsible for the appearance of strains simultaneously virulent and resistant 13 .
In the current study, based on Table 3 and Table 4, statistically significant differences were observed for resistance to the various class of antibiotics and biofilm regulating genes in A. baumannii and P. aeruginosa. Table 4 also showed that a strong relationship between pgaABC and csuABC operons with β-lactamase enzymes (~50% in resistant vs. ~19% in susceptible, p< 0.001). By comparing the results of various studies we determine a significant relationship between β-lactamase enzymes and biofilm operons in A. baumannii 24,27 . However, some researcher showed that there is no significant relationship between biofilm formation and β-lactamase enzymes in P. aeruginosa 22 . This is in contrast to our results, which confirm that the biofilm regulatory gene in P. aeruginosa is most abundant in the β-lactamase producing strains (~35% in resistant vs. ~11% in susceptible, p< 0.001). This contrast in results may be due to differences in the number and type of clinical specimen. In other words, the statistical analysis with these explanations may not be the same in various studies. Therefore, Wang et al. 28 found that there was no significant relationship between antibiotic resistance and biofilm formation in A. baumannii bacteraemic pneumonia.
So far, the knowledge of the relationship between resistance and virulence traits in NFGNBs compared to other bacteria is limited. Environmental factors are one of the most critical factors that can clarify the relationship between biofilm formation (the activity of biofilm operons) and β-lactamase enzymes. Antimicrobial resistance could also be stress-dependent; for example, some studies found that antibiotic resistance increased when subjected to pH (5.0 and 4.0) and salt stresses, although antimicrobial susceptibility returned to previously tested levels after removing the stressors, bacterial sustained antimicrobial resistance 29,30 . This suggests that the pressures of stressors could permanently alter antimicrobial resistance in bacteria; Likewise, these variables can affect biofilm formation. By studying different variables on the activity of biofilm operons and increasing antibiotic resistance, we can determine the effect of environmental factors on the pathogenicity and antibiotic resistance of NFGNBs.
The limitations of the present studies include the academic budget deficit to evaluate the expression level of biofilm regulatory genes in β-lactamase producing strains. Because the expression of biofilm regulatory genes in MDR/ XDR strains may be different with β-lactamase producing strains.

CONClUsiONs
Our results demonstrated that biofilm operons play an essential role in the antibiotic resistance of P. aeruginosa and A. baumannii. In other words, our data show an association between biofilm operons and the abundance of β-lactamase producing strains. Some associations were detected that could help in predicting the degree of virulence of a certain isolate. Nevertheless,this association could be useful for clinicians in terms of adjusting treatment regimens based on the expected degree of virulence and the severity of the illness of the patient. Moreover, this association could be exploited by infection control specialists through the adaptation of eradication protocols to specific isolates. Although the interplay between resistance and biofilm operons seems to be a highly complex one, this observation could suggest that its lack of resistance could be attributing to its increased virulence. Performing a similar study on more sporadic isolates, and isolates from different origins could reveal further clinically important associations and help better understand the interaction between antimicrobial resistance and biofilm operons. Therefore, the identification of biofilm operons in different strains of P. aeruginosa and A. baumannii helps to control antibiotic resistance.