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Specific type IV pili groups in clinical isolates of Pseudomonas aeruginosa

The relationships between specific type IV pili (TFP) groups and antibiotic resistance, biofilm formation, and bacterial motility were determined in 190 Pseudomonas aeruginosa clinical isolates. While motility and biofilm formation were determined by phenotypic assays, the presence of TFP was determined by PCR assay and antibiotic susceptibility by disk diffusion. The results showed a high ability to form biofilm (97.4%), multidrug resistance (44.7%), and the presence of a high number of motile isolates. We also found an association between strong biofilm production and multidrug resistance. Furthermore, TFP group III was associated with strong biofilm production. In contrast, the isolates with TFP group II and those without any TFP were associated with non-strong biofilm production. Regarding motility, TFP group II was associated with higher percentages of swarming, swimming, and twitching, while TFP group I showed lower percentages of swarming and twitching, and TFP group III showed lower levels of swarming and swimming. In conclusion, these findings highlight the differences in P. aeruginosa phenotypes related to the presence of specific TFP groups and their potential implications in clinical settings.


Introduction
Pseudomonas aeruginosa is considered an opportunistic human pathogen being mainly associated with nosocomial infections. P. aeruginosa has the ability to rapidly develop resistance to antibiotics generating multidrug-resistant (MDR) isolates leading to serious problems in hospital settings (Moskowitz et al. 2004).
P. aeruginosa populations undergo frequent recombination events contributing to the evolution of successful epidemic clones (López-Causapé et al. 2017). Thus, while diverse P. aeruginosa populations may be present in different hospital environments (Varin et al. 2017), well-adapted clones may cause nosocomial outbreaks (Oliver et al. 2015).
A biofilm is defined as a community of microbial cells enclosed in an extracellular matrix and associated with a surface with architecture complexity (Deligianni et al. 2010). Despite containing molecules such as DNA or proteins, the extracellular matrix enclosing biofilm cells is predominantly made up of different exopolysaccharides such as alginate, Pel (from "Pellicle"), or Psl (from "Polysaccharide Synthesis Locus" (Mann and Wozniak 2012). The specific relevance of each exopolysaccharide is related to specific bacteria characteristics (Høiby et al. 2017;Mann and Wozniak 2012;Wozniak et al. 2003). Thus, alginate plays a major role in P. aeruginosa mucoid strains such as those prevalent in cystic fibrosis, and Pel and Psl are the most relevant among nonmucoid P. aeruginosa isolates (Høiby et al. 2017;Mann and Wozniak 2012;Wozniak et al. 2003). Biofilm formation causes considerable problems in medical and industrial Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10123-018-00035-3) contains supplementary material, which is available to authorized users. settings, because bacteria in biofilms may be resistant to antibiotic treatment, host immune responses, and biocide treatment (Harmsen et al. 2010). Microscopic analyses have indicated that biofilm formation occurs in a sequential process of (i) transportation of microbes to a surface, (ii) initial attachment, (iii) formation of microcolonies, (iv) biofilm maturation, and (v) biofilm dispersion (Klausen et al. 2003). P. aeruginosa presents three types of motility. Twitching motility, which is mediated by different factors including type IV pili (TFP), allowing dissemination from the initial point of colonization via solid surfaces, interfaces, or moderate viscosities with repetitive alternating movements of extension and retraction (Kazmierczak et al. 2015). In addition, twitching is also involved in biofilm architecture and is responsible for the formation of microcolonies in biofilms (O'Toole and Kolter 1998). Swarming and swimming motilities are mediated by flagella and allow the movement of this microorganism on surfaces and in aqueous environments (Kazmierczak et al. 2015).
The major subunit of TFP is a protein encoded by the pilA gene. This gene is found at a conserved chromosomal locus between the adjacent pilB and tRNA Thr genes (Kus et al. 2004). In addition, pilA has five pili alleles with its accessory genes. Of these, only TFP group II presents the pilA gene without any accessory gene. TFP group I may be divided into subgroups defined by differences in the pili and accessory genes: Thus, subgroup Ia presents the tfpO a gene and Ib possesses the tfpO b gene (Kus et al. 2004). TFP group III and group V pilins possesses the tfpY and tfpZ accessory genes, respectively, and TFP group IV pili have the accessory genes tfpW and tfpX. The pilA alleles belonging to TFP group I (pilA I ) and TFP group II (pilA II ) have shown to be more closely related among themselves than with TFP group III, IV, and V pilins (Asikyan et al. 2008).
Differences in P. aeruginosa phenotypes related to the presence of a specific TFP group remain underexplored, being even less studied in clinical isolates of this microorganism. Therefore, the objective of this study was to determine the relationships between specific TFP groups and antibiotic resistance, biofilm production, and bacterial motility in P. aeruginosa isolates from patients from two hospitals in Lima, Peru.

Bacterial isolates
A total of 190 P. aeruginosa isolates from clinical samples including bronchial secretions (sputum, trachea secretions, and bronchoalveolar lavage), urine, wounds/abscesses, and other (blood, body fluids, catheters, and other unspecified sources) from patients attended at the Hospital Arzobispo Loayza (HAL, 78 strains) and the Hospital Nacional Cayetano Heredia (HNCH, 112 strains) from December 2012 to June 2013 were studied. In all cases, only nonduplicated isolates from different patients were included in the study. The isolates were stored at − 80°C in skim milk medium until use. Each isolate was identified using conventional biochemical tests (Garcia 2010). All samples were obtained within routine clinical practice; no personal data was requested or available to researchers.

Clonal relationships
DNA fingerprinting of all isolates was generated by BOX-PCR as described previously (Mitov et al. 2010). In all cases, the bacterial DNA was extracted by direct boiling (Feizabadi et al. 2010). The BOX-PCR profiles were analyzed according to the similarity of bands calculated by the Dice coefficient using Info Quest software (version 5) (Bio-Rad Laboratories, Inc). Parameters of 1.0% tolerance and 0.5% optimization were used, and similarity matrices were generated with the unweighted pair group method using arithmetic averages (UPGMA). Isolates showing ≥ 85% of similarity were considered to be related.
The isolates were classified as SBP and non-SBP (MBP + WBP + NBP) for statistical purposes. The reference strain P. aeruginosa PAO1 was used as a positive biofilm control.

Motility assays
Swimming, swarming, and twitching motilities were assayed on agar plates containing specific medium according to the methodology of Gupta et al. (2016) and Deligianni et al. (2010). All the plates were inoculated from an overnight culture using a sterile toothpick and were incubated at 37°C for 48 h (Deligianni et al. 2010;Gupta et al. 2016). Motility was determined as the radius of the circular expansion of bacterial growth from the point of inoculation. For swimming and swarming motilities, a measurable zone ≥ 25 mm was considered positive, and twitching was considered as positive when ≥ 10 mm (Otton et al. 2017). The strain PAO1 was included in the analysis as a positive motility control.

TFP detection
The presence of TFP was determined by PCR. The primers used (Table 1) were designed by Kus et al. (2004).

Identification of accessory genes
The primers used for identification of strains containing the tfpO a , tfpO b , tfpW, tfpX, tfpY, and tfpZ accessory genes downstream from pilA were designed for this study by our group (Table 1). The PCR consisted of 15-min denaturation at 95°C followed by 30 cycles of 30 s at 95°C, 1 min at 55°C, and 2 min at 72°C, with a final extension of 7 min at 72°C. The quality of the PCRs was confirmed by the random selection of different amplified products to be sequenced.

Statistical analysis
The χ 2 (chi square) test text was used to determine the presence of significant differences among categorical data, which were considered statistically significant with a p value of ≤ 0.05. Adjustments for multiple comparisons were made using the Holm and Benjamini-Hochberg approaches.
The normal distribution of quantitative data set was established by the Shapiro-Wilk's W test; the one-way ANOVA test and Tukey's post hoc with 95% confidence interval were used to compare differences between the individual TFP groups. R study version 3.4.0 was used for all the statistical analyses.
The associations of isolates possessing TFP group V were not analyzed because of the small number of positive isolates. When not explicitly indicated, TFP groups Ia and Ib were analyzed together as TFP group I.

Bacterial isolates
During the study period, P. aeruginosa isolates were collected from a total of 190 non-duplicated clinical isolates. P. aeruginosa was most frequently isolated from bronchial secretions 37.9% (72/190), urine 29.5% (56/190), and wounds/abscesses 17.4% (33/190). The proportion of isolates recovered from urine samples was significantly (p = 0.0059) higher in HAL (32 isolates, representing 41% of HAL samples and 57.1% of total urine samples) compared to HNCH (24 isolates, representing 21.4% of HNCH samples and 42.9% of total urine samples). Finally, the colony characteristics of 187 isolates were similar to PAO1. Thus, none was mucoid, only three isolates showed morphology of small colony variant (SCV), all isolates but two were β-hemolytic, and 142 isolates were pigmented.

Clonal relationships
The analysis of the clonal relationships by BOX-PCR of the 190 isolates resulted in the identification of 72 clones (Supplementary Table). Of these, 27 (37.5%) were represented by a single isolate, 41 (56.9%) included two to six isolates, and the remaining four clones included more than six isolates. In clones including more than one isolate, a high internal variability in terms of TFP groups, antibiotic resistance, biofilm production, and bacterial motility was observed (Supplementary Table).
In all cases, the SBP isolates exhibited higher levels of antimicrobial resistance than the non-SBP isolates, except in the case of PTZ in HAL. Overall, the SBP isolates were significantly more resistant to CAZ, FEP, ATM, IMI, MER, TO, AK, and CIP (p < 0.05), and accordingly, were associated with the presence of MDR (p = 0.0179) ( Table 2). When biofilm formation was examined according to the clinical origin of the samples, only isolates from wounds/abscesses (78.8% (26/33)) were associated with non-SBP (p = 0.0186).

Type IV pili
Overall, 161/189 (85.2%) of the isolates presented at least one TFP. Of these, 135 (83.9%) presented only one TFP group, and the remaining 26 (16.1%) had more than one TFP group (Tables 3 and 4). TFP group II was the most frequently detected, being found in a total of 103 isolates, being the only TFP group detected in 83 of these isolates (43.9% of the total isolates, 51.5% of isolates possessing TFP). TFP group I was detected in 45 isolates (together with other TFPs in 16 isolates). Group III was detected in 34 isolates (together other TFPs in 14 isolates). Group V was present in 12 isolates, being the only TFP in three isolates. No isolate carrying the TFP group IV was detected ( Table 4).
The absence of TFP (p = 0.0005) as well as the presence of only the TFP group II (p = 0.0260) was significantly higher in the isolates from HAL, while the presence of TFP group I (p = 0.0153) and group III (p = 0.0260) was higher among HNCH isolates. Isolates carrying more than one TFP were also more frequent in HNCH (p = 0.0044).

TFP, motility, and biofilm
Overall, 14.9% (24/161) of the isolates presenting TFP did not show twitching motility, while 92.9% (26/28) of the isolates in which no TFP group was detected did. On evaluating the relationship between the TFP group and flagellar motility (swarming or swimming) it was observed that the presence of only TFP groups I and III were associated with less swarming (p = 0.023/p = 0.024); TFP group III also showed less swimming motility (p = 0.022), and isolates possessing only TFP group I were those with the least twitching motility (p = 0.045) (Fig. 2b, Table 4). The highest levels of twitching were found among the isolates with TFP group II and those without TFP (> 90% in both cases).
Overall, the presence of TFP was associated with SBP (p = 0.02); but when biofilm formation was related to the different TFP groups, only group III showed a significant association with SBP (p = 0.025), while group II (p = 0.025) was associated with non-SBP and those without TFP almost reached significance (Fig. 2c, Table 4). Similarly, the one-way ANOVA test showed a p = 0.058, bordering the significance breakpoint, when TFP contribution to biofilm biomass was determined (Fig. 3a). On analyzing the relationship between TFP groups and biofilm biomass with the one-way ANOVA test, a significant association was observed (p < 0.0001) with groups I, III, and those with more than one TFP presenting greater biofilm biomass (Fig. 3b).
On the other hand, twitching motility was also associated with non-SBP (p = 0.0072) and lower biofilm biomass (p < 0.0001) (Fig. 3g). On analyzing the specific role of each TFP group, irrespective of the type of motility, isolates presenting TFP groups I and III and those with more than one TFP presented greater biofilm biomass, while those with TFP group II or without TFP presented lower biofilm biomass levels (Fig. 3d, f, h). Finally, those isolates without swarming, swimming, or twitching showed higher levels of biofilm biomass (Fig. 4).

Discussion
This study was aimed to determine the relationships between specific TFP groups and antibiotic resistance, biofilm production, and bacterial motility. The prevalence of the different TFP groups observed in the present study is similar to those of other authors in which TFP group I was present in 18% (28/ 159) of human clinical isolates, and TFP group III represented 7% of 244 isolates of P. aeruginosa obtained from a wide  range of environments (Kus et al. 2004;Asikyan et al. 2008). To our knowledge, no specific study has determined the presence of possible associations of specific TFPs with motility, enhanced biofilm formation, or factors favoring the selection of isolates carrying multiple TFP. Our results showed a relation between the number of TFP groups and multidrug resistance. Thus, isolates without TFP showed the lowest levels of multidrug resistance. Nonetheless, although the reason for this is not clear, around 60% of isolates carrying TFP group I or III were MDR while isolates having the TFP group II were more prone to be non-MDR. Further analyses in clinical settings from both Peru and other countries are needed to demonstrate this association providing a rationale to determine the underlying reasons.
The implication of pilA in swarming motility needs to be fully elucidated. Thus, while Shrout et al. (2006) showed swarming motility was not impaired in a pilA deficient strain and, in some circumstances, even showed hyperswarming motility, Köhler et al. (2000) observed the absence of swarming in TFP-deficient P. aeruginosa mutants. The present study showed differences in the association of the TFP groups with swarming and also swimming phenotypes. In agreement with the association of swimming and swarming with flagellar activity, the presence of TFP in our study was not correlated with these motilities, with motile and nonmotile isolates presenting TFP. Nonetheless, our data show that the presence of TFP groups I and III is related to a lower presence of swarming and swimming abilities. This result provides additional information to the previous description of the impairment of swarming motility related to the presence of TFP (Anyan et al. 2014) demonstrating the association of this finding with specific TFP groups. Interestingly, twitching motility was also more frequently present among isolates with TFP group II and among those without TFP. Thus, in our study, 92.8% (26/28) of the isolates not associated with any TFP group were twitching-phenotype positive. While the presence of other adhesins cannot be ruled out (Chiang and Burrows 2003), the presence of polymorphisms on the primers annealing region and that of undescribed TFP possessing new arrangements or longer additional complementary genes leading to long DNA regions which are difficult to amplify with the present PCR conditions and reagents should also be considered. Similarly, the possible insertion of a mobile element before or after TFP genes, which does not affect the functionality of the TFP but does impair PCR amplification, is another possible explanation. On the other hand, isolates possessing TFP but without twitching motility may be explained by the presence of altered or inactive TFP or by the lack of or underexpression of other necessary genes (Chiang and Burrows 2003). This study suggests an association between specific TFP and levels of biofilm formation. In this sense, it was of note that the presence of TFP was significantly associated with SBP, and a p value of 0.058 was observed when the presence of TFP was associated with biomass levels. In this way, the biofilm formation is classified by categories, while biomass is a numerical data obtained from the OD which explains this slight difference. Several studies have reported the importance of bacterial motility to initiate contact with an abiotic surface, biofilm formation, and development (Deligianni et al. 2010;O'Toole et al. 2000). Nonetheless, the associations between the presence of specific TFP groups and biofilm formation ability remain understudied, although they might be related to the absence or presence of TFP accessory genes. In this sense, although it has been shown that a higher twitching level impairs the ability of biofilm formation (Haley et al. 2014), it has been suggested that twitching is one of the factors involved in the first stages of biofilm formation (O'Toole et al. 2000), and consequently, in the degree of biofilm biomass (Deligianni et al. 2010 (Asikyan et al. 2008). Therefore, the presence of accessory genes might positively influence the biofilm formation. Our results agree with the impairment of biofilm formation related to the presence of twitching (Haley et al. 2014), and isolates presenting twitching are correlated with lower levels of biofilm biomass. Furthermore, the higher levels of biomass biofilms were correlated with the absence of swimming, swarming, and twitching, while isolates presenting the three types of motility were those with lower biofilm biomass. This result highlights the fact that isolates with lower motility levels are more prone to adhere to surfaces leading to biofilm formation. Nevertheless, contrary to the proposed correlation between TFP accessory genes and enhanced twitching (Asikyan et al. 2008), in all the isolates presenting TFP group II, the only TFP group lacking accessory genes, or without any TFP showed higher levels of motility. On the other hand, the presence of isolates without TFP classified as SBP and that of isolates with a very poor adhesion to the microtiter plate despite possessing TFP (including TFP group III) agree with the fact that mutants deficient in pili and flagella do not exhibit significant differences regarding biofilm formation compared to wild-type strains (Klausen et al. 2003). Therefore, these findings point to the possible role of other new adhesins that could participate in the initial stage of biofilm formation (Otton et al. 2017;Head and Yu 2004) and highlight the multifactorial nature of biofilm formation.
In a recent study, the development of quinolone resistance was correlated with lower levels of twitching activity (Ahmed et al. 2018). Thus, as our results showed that isolates presenting lower motility levels were related to SBP, this finding may be related to the high levels of antibiotic resistance detected in those isolates being SBP. Accordingly, all isolates deficient for the three tested motilities were SBP and MDR, and the presence of multidrug resistance was lesser among those isolates possessing TFP group II or those without TFP which showed high motility levels.
Our results showed the presence of differences in the prevalence of TFP as well as of TFP groups and other characteristics among the samples from the two hospitals. This finding suggests the presence of differences among both bacterial populations. Nonetheless, no specific reason may be adduced.
The intraclonal variability of the parameters analyzed must be considered among limitations of this study, since it prevents analysis corrected by phylogeny. This variability may be related to horizontal gain or loss of genetic material or with undetected punctual mutations, insertions, or deletions which affect the final expression levels of key genes.
Overall, the presence of TFP was correlated with enhanced biofilm formation; furthermore, the presence of specific TFP groups was correlated with different findings, including MDR (group III) or non-MDR (group II) profiles and levels of swarming/swimming/twitching motility (group I and group III with lower levels of motility, group II or those without TFP with higher levels of motility). The present results suggest that isolates with impaired motility are more prone to being SBP. These findings highlight the need for an in-depth analysis of the underlying differences and elucidation of the exact relationships between specific TFP groups, biofilm production, and the acquisition of multidrug resistance. Fig. 4 Box plots showing the association of motility with biofilm biomass. For swimming and swarming motilities, a measurable zone ≥ 25 mm was considered positive, and twitching was considered as positive when ≥ 10 mm (Otton et al. 2017). Absence, Lack of swarming, swimming, and twitching; Presence, concomitant presence of swarming, swimming, and twitching; Mixture, presence of a minimum of one motility type and a maximum of two motility types. When the different combinations of "Mixture" motility group were analyzed individually, no differences were observed among them