Phenotypic and genetic analysis of virulence factors in multidrug- sensitive and multidrug-resistant clinical isolates of Pseudomonas aeruginosa

This study aimed to correlate the pattern of antimicrobial susceptibility, phenotypic production of virulence factors, the occurrence of virulence factors genes and the clonal profile of clinical isolates of Pseudomonas aeruginosa of a tertiary hospital in Recife-PE. The 30 clinical isolates (15 multidrug-sensitive (MDS) and 15 multidrug-resistant (MDR)) were analyzed using phenotypic methods to detect virulence factors (alkaline protease, hemolysin, phospholipase C, lipase, and pigments). The detection of the aprA, lasA, lasB, plcH, and toxA genes was performed through specific PCRs, and the clonal profile was assessed using ERIC-PCR. The results revealed cephalosporins being the class eliciting the highest percentage of resistance; the MDR isolates were all resistant. Among the MDS isolates, all were sensitive to carbapenems and quinolones. The MDR isolates produced less virulence factors such as pyocyanin and lipase, and exhibited lower expression of toxA and lasA genes, whereas the MDS isolates produced less hemolysin and phospholipase C. There was no difference between the groups for alkaline protease production and aprA gene expression. All the isolates produced pyocyanin and expressed lasB and plcH genes. A great genetic diversity was found, and it was possible to observe 28 genetic profiles. Clones were present among the MDR isolates. The occurrence of virulence factors in almost all the isolates studied suggests their high level of pathogenicity, demonstrating that this pathogen is capable of accumulating numerous virulence factors, and in some cases, is associated with multidrug resistance, which makes it difficult to treat these infections.


Introduction
Pseudomonas aeruginosa, one of the main pathogens involved in hospital infections of immunocompromised patients, causes the most frequent healthcare-related infections associated with drug-resistant bacteria. This bacterium is considered an opportunistic infectious agent and exhibits several pathogenic mechanisms as well as resistance to various antimicrobials (Paz-Zarza et al., 2019).
It is one of the six pathogens in the ESKAPE group (comprising Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter spp., P. aeruginosa, and Enterobacter spp.) and one of the main causes of healthcarerelated infections in the United States and worldwide, owing to its ability to become resistant to available antimicrobials (Lupo, Haenni & Madec, 2018).
Its mechanisms of resistance to antimicrobials can be intrinsic or acquired, and are related to therapeutic failure for infections related to it (Silva Júnior et al., 2017). Combinations of these resistance mechanisms have given rise to multidrugresistant (MDR) isolates (Magiorakos et al., 2012). Adding to the lack of perspectives for the introduction of new drugs that could be used against this pathogen, such evolutions provided the need for investigations on its physiology, mainly with regard to its diverse virulence factors that also contribute to the pathogenic potential of the infectious process, since they favor Research, Society andDevelopment, v. 10, n. 11, e457101120032, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.20032 3 infection by increasing tissue damage and protecting P. aeruginosa against recognition by the immune system and the action of antimicrobials (Todar, 2020).
Moreover, some studies have shown a high percentage of virulence factors in isolates of P. aeruginosa. In a study carried out in China (Wang et al., 2013), approximately 80% of the isolates studied produced elastase, pyocyanin, and alkaline protease. In Brazil, Jácome et al. (2012) got similar results (34.4% mucous colonies, 70.5% pyocyanin producers, 93.4% gelatinase producers, and 72.1% hemolysin producers); in relation to the resistance pattern, 54.1% of the isolates were MDR and 4.9% were pandrug-resistant.
A study carried out in Poland by Deptuła & Gospodarek (2010) revealed that MDR strains produced lower lipase, elastase, phospholipase C, and pyocyanin amounts compared to multidrug-sensitive (MDS) strains, and that there were no significant differences in alkaline protease activity, indicating that P. aeruginosa MDR strains were less virulent than MDS strains. However, the study was limited to phenotypic research of these virulence factors, and the genes responsible for their production have not been confirmed. It is also important to note that the study was carried out with isolates from a single hospital in Poland; taking into account the diversity of P. aeruginosa profiles found in different regions, local studies are necessary.
Therefore, this study aimed to describe the correlation between the presence of virulence factors, through phenotypic analysis of alkaline protease, lipase, phospholipase C, hemolysin, and pigments; occurrence of aprA, lasA, lasB, plcH, and toxA genes; pattern of resistance to antimicrobials (multidrug sensitivity or resistance); genetic variability; and origin of clinical isolates of P. aeruginosa.

Bacterial Isolates
The number of isolates for this study was convenience sample thus thirty bacterial isolates of P. aeruginosa from samples obtained at public hospitals in Recife, Pernambuco, were analyzed. The isolates were biochemically identified, subjected to antimicrobial susceptibility tests that were evaluated using the automated BD Phoenix system (CLSI, 2015), and frozen in 20% glycerol at −20 °C in the bacteriology library of the Bacteriology and Molecular Biology Laboratory of the Department of Tropical Medicine of the Federal University of Pernambuco (UFPE). The isolates were selected by nonprobabilistic, according to their susceptibility profiles and classified according to CLSI (2015) into MDS and MDR, with a total of 15 isolates per group.
For analysis, the isolates were reactivated after inoculation in Brain Heart Infusion (BHI) broth and incubated for 24 to 48 h in an oven at 37 °C. After bacterial growth, the isolates were seeded on cetrimide agar, which is selective for the isolation of P. aeruginosa, and were also cultivated on nutrient agar to conserve them in stock.

Pigment Production
Pigment (pyocyanin, pyoverdine, pyorrubin and pyomelanin) production was evaluated by observing the color corresponding to each pigment presented in the culture medium (cetrimide agar) after 24 h of incubation at 37 °C. Moreover, the production of pyoverdine was confirmed when the cultures emitted fluorescence upon exposure to ultraviolet light (Winn Jr et al., 2008).

Hemolysin Production
To evaluate hemolysin production, isolates were seeded on blood agar and incubated for 24 h at 37 °C. The isolates with a translucent halo around the colonies, indicating hemolysis, were considered hemolysin producers (Winn Jr et al., 2008).

Alkaline Protease Production
Alkaline protease production research was carried out according to the Jagger, Bahner & Warren (1983) method: Isolates were inoculated with skim milk agar (2%) and incubated at 37 °C for 24 h. The presence of a translucent halo around the colonies was interpreted as a positive result, and the absence of this halo as a negative result. The reference strain P.
aeruginosa ATCC 15692 (PA01) was used as a positive control.

Phospholipase C Production
To determine phospholipase C production, the method described by Habermann & Hardt (1972) was used, with modifications. Isolates were inoculated in spots on plates containing trypticase soy agar (TSA) enriched with 10% (vol/vol) egg yolk solution with tellurite. The plates were then incubated in an oven for 24 h at 37 °C. The production of a black precipitate in the growth zone signaled a positive result for the production of phospholipase C. The reference strain P.
aeruginosa ATCC 15692 (PA01) was used as a positive control.

Lipase Production
Lipase production was determined according to the method described by Janda & Bottone (1981). Isolates were inoculated in TSA enriched with 1% Tween 80 and incubated at 37 °C for 24 h. The formation of a cloudy halo around the growth zone was considered positive for the production of lipases, and the absence was considered negative. The reference strain P. aeruginosa ATCC 15692 (PA01) was used as a positive control.
Gene Virulence Detection PCR reactions were performed as described by Lanotte et al (2004) and Lomholt, Poulsen & Kilian (2001) with modifications. The primers are described in Table 1. The amplification reactions were performed in a final volume of 25 µL containing 10 ng of DNA, 0.2 mM of dNTP, 20 pmol of each primer, 2 mM MgCl2, and 1.0 U Taq DNA polymerase in 5× reaction buffer. The DNA was amplified in a thermocycler using the following protocol: initial denaturation at 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min, and extension at 72 °C for 1 min 30 s, and then a final extension at 72 °C for 5 min. Each gene was amplified separately. An exception to the described protocol was made for the aprA gene, for which a ring temperature of 62 °C was used. The PCR products were subjected to electrophoresis in 1% agarose gel, stained with blue-green dye (0.05 µg/mL) at 100 V for 30 min, detected by UV transillumination, and photo-documented.
Pseudomonas aeruginosa strain ATCC 15692 (PA01) was used as a positive control.

Bacterial Isolates
Related the origin of the sample of the bacterial isolates, 50% the source was tracheal secretions, 20% from urine and blood, with the catheter tip being the site of least origin from the isolates, 7%.

Analysis of the profile of susceptibility to Antimicrobials
The data of the susceptibility profile are shown in Figure 1.  Research, Society and Development, v. 10, n. 11, e457101120032, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org /10.33448/rsd-v10i11.20032 6 The frequency of resistance to the antimicrobial classes between MDS and MDR isolates is shown in Figure 2, and the distribution of the frequency of the isolates according to the number of classes to which they were resistant is described in Table 2. It is noted that among MDS isolates there was a higher frequency of isolates resistant to cephalosporin 66.67% (10/15) and all isolates tested were sensitive to carbapenems and quinolones, and among MDR isolates all isolates showed resistance to the cephalosporin class, and a lower frequency of resistance to aminoglycosides 53.34% (8/15).

Phenotypic detection of virulence factors
Regarding the phenotypic search for virulence factors pyocyanin, pyoverdine, pyomelanin, hemolysin, protease, lipase and phospholipase C, the results are described in nominal form (present/absent of the virulence factor), and are shown in Figure 3. There was a higher production of phospholipase among MDR isolates and related the MDS isolates there was a higher production of lipase.
The production of bacterial pigment pyorubin was not observed in any of the groups analyzed. Pyomelanin was observed in sensitive isolates, but in a low percentage, and pyocyanin pigment was present in sensitive and antimicrobial resistant isolates, and in relation to pyoverdine it was produced in both groups, this pigment being described in 100% of the isolates. The difference between the production of hemolysin in the two groups it was low and protease factor were frequently elevated in both groups. Source: Authors.

Virulence Genes Distribution
The results in Figure 4 show the frequency distribution of virulence genes according to the resistance pattern. The MDS isolates obtained a 20% higher frequency of the toxA and lasA genes, than the MDR isolates, all isolates in the study had the lasB and plcH genes. Source: Authors.

Genetic Relation
The molecular typing of the isolates revealed 28 genetic profiles, with the occurrence of two isolates showing a clonal relationship, Figure 5, is the representation of the genetic similarity dendrogram among all P. aeruginosa isolates estimated by the ERIC-PCR technique. Source: Authors.
The Figures 6 A and B show dendrograms of genetic similarity between MDS and MDR isolates, respectively, where it is possible to see that the mentioned clones were found among MDR isolates.

Discussion
In this study, there was high resistance for cephalosporins among the isolates of P. aeruginosa studied. These antimicrobials are used in the treatment of serious infections, such as septicemia caused by this pathogen (Pérez et al., 2012).
The tendency toward less sensitivity to cephalosporins has been described in other regions of Brazil; this might be due to the large-scale use of this drug as a therapeutic option, for many years, for patients with various infections in public hospitals (Jácome et al., 2016;Siqueira et al., 2018;Furtado et al., 2019;Ribeiro et al., 2020).
The isolates in these studies had low resistance to carbapenems; however, with temporal evolution, this frequency has increased, as described by Jácome et al. (2016) and Lima et al. (2020), reaching 53% and 40%, respectively, among hospital isolates from Recife-PE. The MDR isolates exhibited a lower frequency of resistance to aminoglycosides; this has also been verified in the studies of Siqueira et al. (2018) and Montoro et al. (2019). The great diversity presented in the susceptibility profile to the antimicrobial classes suggests the existence of an association of several resistance mechanisms, which was beyond the scope of this study.
The MDS/MDR isolates analyzed in this study were most prevalent in tracheal secretions of patients admitted to Intensive Care Units (ICUs). This result is not surprising, since P. aeruginosa is one of the main microorganisms related to infections in patients having pneumonia and requiring mechanical ventilation (Sader et al., 2015).
An analysis of the production of virulence factors among the isolates in this study indicated a higher production of pyocyanin in the MDS isolates, favoring pathogenesis by these microorganisms; this was also observed by Deptuła and Gospodarek (2010) and El-Mahdy and El-Kannishy (2019). Overexpression of pyocyanin was detected in different samples of airway secretions from patients colonized by P. aeruginosa and was associated with the severity of the disease and a decrease in lung function (Rada & Leto, 2013). Silva et al. (2014) on the other hand, described an increase in the production of pyocyanin in isolates from urinary tract infections.
Related of production of pigments and other virulence factors in isolates of P. aeruginosa, the study by Finlayson and Brown (2011), described that in the pigmented (production of pyocyanin and/or pyoverdin) isolates of P. aeruginosa from different clinical samples there was more production of elastase, protease and lipase than non-pigmented isolates, in the present study, all the isolates MDR or MDR presented some type of pigment, which may be related with a high frequency of other virulence factors.
Hemolysin production was high among the isolates of both groups. Hemolytic phospholipase C and ramnolipids are hemolysins produced by P. aeruginosa (Ostroff, Vasil & Vasil, 1990). In this study, the plcH gene was detected in all the isolates, however, nine isolates did not show phenotypic production of phospholipase C, which may have been due to the regulation of gene expression (Heidary et al., 2016).
Furthermore, proteases, another type of virulence factor produced by P. aeruginosa that helps in its pathogenesis, were widely detected among MDS and MDR isolates. These data corroborate those presented by Deptuła and Gospodarek (2010), who observed no difference between the groups analyzed. Shigematsu et al. (2001) carried out in vitro studies on P.
aeruginosa and demonstrated that the protease enzyme is expressed in environments with free-iron deficiency, is important in the acquisition of iron at the site of the infectious process and favors bacterial virulence. We found two isolates that did not show proteolytic activity in the phenotypic test and were negative for the aprA gene.
Most of the isolates in this study secreted the enzyme elastase B (LasB), an elastolytic metalloproteinase that is encoded by the lasB gene (Nikbin et al., 2012). In addition to damaging tissues, LasB is capable of degrading components of the innate and acquired immune system, including cytokines and antimicrobial peptides (Kuang et al., 2011). In this study, lasA and lasB genes were detected at a lower frequency among MDR isolates compared to MDS isolates, corroborating the data of Deptuła and Gospodare (2010) and El-Mahdy and El-Kannishy (2019), however, it is necessary to remember that both lasA and lasB are regulated by quorum sensing, and hence, that their expression varies greatly depending on the regulation.
Another gene with higher occurrence among MDS isolates was the toxA gene, which is a structural component of exotoxin A. This enzyme is a determinant virulence factor of P. aeruginosa and is considered the most toxic and potent among other virulence mechanisms (Hassuna, Mandour & Mohamed, 2020). Some reports state that there is a relationship between lasB and toxA genes in strains isolated from the lungs of patients with cystic fibrosis, in which the transcripts of these genes are regulated more in bacterial cells (Storey et al., 1997). Moreover, LasB is a well-studied virulence factor that minimizes the immune response and is the target of alveolar macrophages (Andrejko et al., 2013).
The results obtained in this study demonstrated a correlation only for some of the studied virulence factors, suggesting that this relationship is multifactorial. The MDR isolates produced less pyocyanin and lipase, and exhibited lower expression of the toxA and lasA genes, whereas the MDS isolates produced less hemolysin and phospholipase C. It has been reported that the acquisition of virulence factors by MDR bacteria during the infectious process can lead to unnecessary metabolic costs for the microorganism (Sanchez et al., 2002) and that the addition of resistance mechanisms leads to a decrease in virulence factors (Deptuła & Gospodarek, 2010). This relationship between virulence and resistance in P. aeruginosa has not been established in this study, some authors as Al Dawodeyah et al. (2018) indicated that there is association between antimicrobial resistance and virulence genes in MDR strains and Persyn et al., 2019, refers this association is poorly understood.
The genetic profile in this study showed a great diversity; with a total of 30 isolates, it was possible to observe 28 genetic profiles. Clones were present among the MDR isolates; interestingly, the clones, P137HC and P109HC, showed discordant results in the test of hemolysin production, whereas P30HC and P45HC showed discordant results for the production of phospholipase C. These results may have resulted from the regulation of gene expression, which was beyond the scope of this study, since P. aeruginosa has quorum sensing systems that are responsible for regulating the expression of plcH (hemolytic phospholipase C), ramnolipids, and other virulence factors, which would explain such phenotypic differences (Strateva & Mitov, 2011).
The presence of these patterns of virulence in almost all the isolates studied highlights the high level of pathogenicity of isolates derived from nosocomial infections. Therefore, P. aeruginosa is a pathogen capable of accumulating numerous virulence factors, and in some cases, is associated with multidrug resistance, which makes it difficult to treat infections caused by it.

Conclusion
In conclusion, a tendency described by several authors, that is, a decrease in virulence factors with an increase in resistance to antimicrobials, was observed in relation to the toxA and lasA genes and in the production of lipase and pyocyanin.
This shows that the acquisition of virulence factors can lead to unnecessary metabolic expenditure for MDR isolates.