Characterization of β-lactamase and virulence genes in Pseudomonas aeruginosa isolated from clinical, environmental and poultry sources in Bangladesh

The emergence and spread of multidrug-resistant pathogens like Pseudomonas aeruginosa are major concerns for public health worldwide. This study aimed to investigate the prevalence of circulating P. aeruginosa isolated from clinical, environmental and poultry sources in Bangladesh, their antibiotic susceptibility, β-lactamase and virulence gene profiling using standard molecular and microbiology techniques. We collected 110 samples from five different locations, viz., BAU residential area (BAURA; n=15), BAU Healthcare Center (BAUHCC; n = 20), BAU Veterinary Teaching Hospital (BAUVTH; n=22), Poultry Market (PM; n=30) and Mymensingh Medical College Hospital (MCCH; n=23). After overnight enrichment in nutrient broth, 89 probable Pseudomonas isolates (80.90%) were screened through selective culture, gram-staining and biochemical tests. Using genus- and species-specific PCR, we confirmed 22 isolates (20.0%) as P. aeruginosa from these samples. Antibiogram profiling revealed that 100.0% P. aeruginosa isolates (n = 22) were multidrug-resistant isolates, showing resistance against Doripenem, Penicillin, Ceftazidime, Cefepime, and Imipenem. Furthermore, resistance to aztreonam was observed in 95.45% isolates. However, P. aeruginosa isolates showed a varying degree of sensitivity against Amikacin, Gentamicin, and Ciprofloxacin. The blaTEM gene was detected in 86.0% isolates, while blaCMY, blaSHV and blaOXA, were detected in 27.0%, 18.0% and 5.0% of the P. aeruginosa isolates, respectively. The algD gene was detected in 32.0% isolates, whereas lasB and exoA genes were identified in 9.0% and 5.0% P. aeruginosa isolates. However, none of the P. aeruginosa isolates harbored exoS gene. Thus, this study provides novel and important data on the resistance and virulence of P. aeruginosa currently circulating in clinical, environmental and poultry environment of Bangladesh. These data provide important insights into the emergence of β-lactamase resistance in P. aeruginosa, highlighting its usefulness in the treatment and control of P. aeruginosa infections in both humans and animals.


Introduction
Pseudomonas aeruginosa, a Gram-negative bacterium, has gained prominence as an emerging opportunistic pathogen with major clinical consequences, particularly in hospital settings where it poses a large risk to immunocompromised persons [1,2].P. aeruginosa, known for its adaptability and metabolic diversity, is a prominent cause of nosocomial infections, producing severe acute and chronic illnesses in individuals with a variety of vulnerabilities [3,4].Its ubiquity goes beyond healthcare facilities since it thrives in a variety of habitats, including soil, water, and even oil-polluted environments, giving it the term "ubiquitous" or "common soil and water bacterium" [5].
Antimicrobial resistance (AMR) in P. aeruginosa, is a significant concern in healthcare settings due to its ability to cause severe infections, particularly in individuals with weakened immune systems.P. aeruginosa is inherently resistant to many antibiotics and has a remarkable capability to develop further resistance mechanisms [6].The increasing fear of P. aeruginosa is exacerbated by the worldwide problem of antimicrobial resistance (AMR), which has been dubbed a "crisis of the twenty-first century" [7].Antibiotic overuse has developed multidrug resistance among bacteria, such as P. aeruginosa, posing a severe danger to public health and economies, particularly in low and middle-income countries (LMICs) in Africa and Asia, including Bangladesh.The growth in antibiotic-resistant P. aeruginosa strains, with over 10% of worldwide isolates demonstrating multidrug resistance, adds to the difficulties in treating infections caused by this bacterium [8].Notably, P. aeruginosa uses a variety of resistance mechanisms, including gene expression under stress and the production of antibiotic-resistant biofilms, which reduces the efficiency of traditional therapies [9].Antibiotic-resistant P. aeruginosa is found in various habitats, including wastewater and soils, emphasizing the necessity of studying the frequency and resistance patterns of these clinically relevant bacteria in various contexts [10].The recent development of extended-spectrum beta-lactamases (ESBLs) complicates the clinical situation even more, with ESBLs being a major contributor to bacterial resistance worldwide [11].As a result of the complications imposed by ESBLproducing isolates, microbiologists, medics, and scientists working on creating novel antimicrobial medications face extra hurdles [11,12].P. aeruginosa is known for its diverse array of virulence factors, contributing significantly to its pathogenicity and ability to cause infections.A variety of virulence factors have been discovered in P. aeruginosa that can significantly contribute to the pathogenicity of this bacterium [4,13].Toxin A (toxA), exotoxin A (exoA), alkaline protease (aprA), elastase, and exoenzymes (S, U, and T exoS, exoU, exoT) are the principal virulence factors.LasB, a zinc metalloprotease, exhibits elastolytic activity specifically on lung tissue of human [14].Chronic lung infections are significantly influenced by the algD gene, which is accountable for the alginate capsule of P. aeruginosa [15].In addition, exotoxin A is secreted via Type II secretion systems, thereby contributing to the extracellular pathogenicity of the bacterium [16].The presence and interplay of these virulence factors in P. aeruginosa contribute to its pathogenicity, enabling it to cause a wide range of infections, particularly in immunocompromised individuals or those with underlying health conditions.Understanding these virulence mechanisms is crucial for developing targeted strategies to combat P. aeruginosa infections and improve patient outcomes [4].
While most P. aeruginosa research in Bangladesh has concentrated on antibiotic resistance profiles in clinical and wastewater isolates, but a thorough examination of environmental isolates in the specified region is lacking.Thus, this study aimed to provide significant insights into the prevalence, antimicrobial resistance, and virulence profiles of multidrug-resistant P. aeruginosa isolates from clinical, environmental and poultry sources in some selected areas of Bangladesh.The findings of this study can significantly contribute to our knowledge of the dynamics of P. aeruginosa, which in turn can inform public health policies, clinical practices, and future research efforts aimed at controlling infections caused by this pathogen.

Isolation and identification of P. aeruginosa
This cross-sectional study was conducted in the Bacteriology Laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh during November 2022 to July 2023.The studied samples (Table S1 S1).Therefore, these samples belonged to three major categories including non-hospital environmental samples (BAURA; 15 samples), hospital-based clinical samples (BAUHCC, BAUVTH and MMHC; 65 samples) and poultry samples (PM, 30 samples).These samples comprising drain water, sewage, soil, and samples from hospital environments and poultry markets (Table S1), were individually placed in sterile test tubes with 3 -4 mL nutrient broth, labelled and transported to the laboratory.A single loopful of overnight culture grown in nutrient broth was streaked over cetrimide agar (HiMedia, India) and aerobically incubated overnight at 37°C.Colonies with distinguishing characteristics, such as greenish color, were assumed to be Pseudomonas genus (n = 89 isolates).The genus-specific identification of P. aeruginosa typically involved a series of standard bacteriological methods including gram staining and an array of biochemical assays, including sugar fermentation, Voges-Proskauer, indole, and catalase tests [4,17].

Molecular identification of P. aeruginosa
Pseudomonas isolates (n = 89) were then molecularly identified using genus-and speciesspecific polymerase chain reaction (PCR) assays.Genomic DNA from overnight culture by boiled DNA extraction method using commercial DNA extraction kit, QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany).Quality and quantity of the extracted DNA were measured using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA).DNA extracts with A260/280 and A260/230 ratios of ∼ 1.80 and 2.00 to 2.20, respectively, were considered as high-purity DNA samples [18] and stored at -20°C prior to PCR amplification [19,20].To amplify the 16S rRNA sequences of Pseudomonas, a set previously designed primer set was used (Table 1).PCR amplification of 16s_ Pseudo for the detection of Pseudomonas genus [21] and Pseudo_aeru for the detection of P. aeruginosa species [22,23] was performed on all phenotypically identified isolates of P. aeruginosa.The PCR condition against these primer sets for the amplification of genus-and species-specific primers is shown in Table S2.Amplification of targeted DNA was carried out in a 20 µL reaction mixture, including 3 µL nuclease-free water, 10 µL 2X master mixture (Promega, Madison, WI, USA), 1 µL forward and reverse primers (for each) and 5 µL DNA template.PCR-positive controls consisted of P. aeruginosa genomic DNA previously confirmed for the target genes [21][22][23].
PCR-negative controls utilized non-template controls with PBS instead of genomic DNA.
Finally, amplified PCR products underwent electrophoresis on a 1.5% agarose gel and visualized using an ultraviolet transilluminator (Biometra, Gottingen, Germany).A 100 bp DNA ladder (Promega, Madison, WI, USA) was used to validate the expected sizes of the amplified PCR products [24].Finally, 22 isolates were confirmed as P. aeruginosa through species-specific PCR.

Molecular detection of antibiotic resistant and virulence genes in P. aeruginosa
For the detection of the antibiotic resistant and virulence genes in the P. aeruginosa isolates, simplex PCR assays for beta-lactam antibiotics resistant genes (e.g., blaTEM, blaCMY, blaSHV, blaOXA), and virulent genes including exoA, exoS, lasB, and algD were performed with specific primers (Table 1).The PCR protocols utilized for detecting these genes were consistent with those described earlier in Section 2.2.

Statistical analysis
Data were entered into Microsoft Excel 2020 ® (Microsoft Corp., Redmond, WA, USA) and analyzed using Excel and SPSS version 20 (IBM Corp., Armonk, NY, USA).The Pearson's chi-square test was performed to compare the prevalence of P. aeruginosa in three different sample categories (e.g., clinical, environment and poultry).A prevalence percentage was calculated by dividing the number of positive samples for the given category by the total samples tested within that category [29,30].The prevalence formula was applied for determining prevalence percentage of Pseudomonas and P. aeruginosa.The AMR patterns, resistance, intermediate and sensitivity, and MAR index were calculated using the CLSI (2021) guideline using the cut-off as provided in the brochure of the manufacturer (Liofilchem ® , Italy).
Correlation coefficients for any of the two resistant antibiotics, association between phenotypic and genotypic resistance patterns, and phenotypic/genotypic resistance patterns and virulence genes of isolated P. aeruginosa was calculated using Pearson correlation tests.For the test, p < 0.05 was considered statistically significant.
Bivariate analysis exhibited that the hospital-based clinical (BAUHCC, BAUVTH and MMCH) samples had a significant correlation with both non-hospital environment samples and poultry market (PM) samples (Pearson correlation coefficient, ρ = 1; p <0.001) (Table S3).

Antibiogram profile of P. aeruginosa
The overall antibiogram profiles of isolated P. aeruginosa are presented in Fig. found to carry a single resistance gene, blaTEM (Fig. 2).By comparing the phenotypic and genotypic resistance pattern of the P. aeruginosa isolates, we found that the Levofloxacin and Gentamycin resistant isolates had significantly higher amount of blaCMY and blaOXA than the other two checked genes (blaTEM and blaSHV) (Table 4).

Virulence genes in P. aeruginosa
Gene-specific simplex PCR was used to screen the virulence genes in 22 P. aeruginosa isolates.
Among aeruginosa isolates, we found no significant association (Tables S4 and S5).

Discussion
Pseudomonas aeruginosa is considered a significant cause of nosocomial or healthcareassociated infections (HAI) and is classified as a priority critical pathogen of global concern by various health organizations [31].As an opportunistic bacterial pathogen, P. aeruginosa has been linked with a range of human and animal infections [32].P. aeruginosa is ubiquitous in the environment (soil and water) and capable to produce disease in individuals with weakened or compromised immune systems [33].Despite breakthroughs in medical and surgical treatment, as well as the introduction of a wide range of antimicrobial drugs in livestock and agricultural, the number of resistant isolates in outdoor surface water has increased considerably [34].Reports from different countries are available describing the detection of P.
aeruginosa from various environmental sources [4,5,33,35].However, sufficient information is unavailable on the environmental distribution of potential pathogenic MDR P. aeruginosa in Bangladesh.Previous studies in Bangladesh have focused on clinical isolates, healthcare workers' mobile phones, and even the genomic diversity of MDR P. aeruginosa from burn patients [4,36,37].Therefore, this study was designed to determine the antibiotic resistance profiles and virulence genes distribution in P. aeruginosa circulating in different environments of Bangladesh.We analyzed 110 samples belonged to three major categories e.g., non-hospital environment (BAURA), hospital-based clinical (BAUHCC, BAUVTH and MMHC) and poultry market (PM) from five distinct locations of Mymensingh division of Bangladesh.These diverse study areas collectively provided a comprehensive overview of the prevalence and distribution of MDR P. aeruginosa and its virulence profiles within distinct environmental contexts, contributing to a holistic understanding of the potential public health significance of these bacteria in the selected region.The overall prevalence of P. aeruginosa was 20.0%.
However, based on locations and sample categories, 26.67%, 13.85%, and 30.0%were reported in non-hospital environment samples (drain water and drain sewages) of BAURA, clinical samples (BAUHCC, BAUVTH and MMCH) and poultry (PM) samples, respectively.The highest isolation rates of P. aeruginosa (30.0%) was found in the PM samples.In Bangladesh, the average isolation rate for P. aeruginosa from diverse surface waters was reported to be 61.5%, which is greater than in its neighboring nation, India (45.45%) [38].The isolation rate of various water samples may fluctuate depending on the source and size of the sample, as well as the time and length of sample collection [3,35,39].Previously, Algammal et al. reported 28.3% prevalence of P. aeruginosa in broiler chickens in Egypt [40] whereas El-Tawab et al. found that 34% of the tested frozen fish possessed P. aeruginosa [41].
Detection of P. aeruginosa in poultry and associated environment is alarming because of the possibilities of transmission of these pathogens to human.
In this study, the most commonly used antibacterial drugs in Bangladesh were tested against P. aeruginosa isolates to determine their resistance pattern.We found that 100% P. aeruginosa isolates were resistant to multiple antibiotics such as Penicillin, Ceftazidime, Cefepime, Doripenem and Imipenem.In addition, 95.45% isolates were resistant to Aztreonam.However, the study isolates showed high to moderate level (44.0 to 78.0%) sensitivity against Amikacin, Gentamicin and Ciprofloxacin.These results corroborated with several earlier studies that investigated antimicrobial susceptibility profile in P. aeruginosa isolated from clinical samples [4], environmental samples [37] and poultry samples [42].Consistent findings across various studies examining the antimicrobial susceptibility profile of P. aeruginosa isolated from diverse sources (clinical, environmental, and animal samples) can lend support to the understanding of the AMR patterns and prevalence of MDR strains within this bacterium.Such cumulative evidence aids in comprehending the overall resistance landscape and guides the development of effective strategies for managing infections caused by P. aeruginosa.We found that 100.0%isolates of the P. aeruginosa were MDR isolates (resistant to > 5 antibiotics).
Furthermore, the multiple antibiotic resistance (MAR) indices for the P. aeruginosa isolates ranged from 0.5 to 0.9 indicating an increased likelihood of these isolates being exposed to various antibiotics or antimicrobial agents, potentially contributing to the development of MDR, posing significant concerns for public health due to the challenges in treating infections caused by these organisms.Indeed, a MAR index greater than 0.2 is indicative of potential contamination from sources where antibiotics are frequently utilized [43].
We also investigated the occurrence of several ARGs which could aid in designing effective treatment strategies and surveillance programs to control the spread of antibiotic-resistant strains of P. aeruginosa.In this study, blaTEM gene was found to be predominating in 86.36% isolates.This high frequency of blaTEM is however, consistent with the findings of Hosu et al.
who reported a 79.3% prevalence of blaTEM in clinical P. aeruginosa isolates, indicating its widespread dispersion in healthcare settings [11,44].the second most prevalent ARG was blaCMY which was detected in about 27.0% isolates.Recently, Ejikeugwu et al. reported that around 42.0% P. aeruginosa isolates from clinical samples harbored blaCMY gene [45], which is a bit higher than our findings.This observation is incongruent with the findings of Mohamed et al. who detected a far higher incidence of blaCMY in P. aeruginosa isolates derived from chickens, indicating possible sources of resistance in non-clinical settings [46].Although, blaSHV, and blaOXA were identified as the less abundant ARG in P. aeruginosa isolates (18.0% and 4.55%, respectively), these ARGs were found in different combination.The detection of blaSHV, and blaOXA genes in P. aeruginosa isolates aligns with findings from previous studies [4,47,48], further highlighting the significance of these genes in healthcare settings and their association with nosocomial infections.Their presence underscores the potential for these bacteria to cause severe infections and emphasizes the need for stringent infection control measures and judicious use of antibiotics to mitigate the emergence and spread of multidrug-resistant strains The difference between the results of this study and other investigations might be attributed to differences in various circumstances comprising incubators or to commonly occurring hyper-mutation among P. aeruginosa strains exhibiting varied antibiotic resistance.Furthermore, antibiotic-resistant bacteria can swiftly spread across the food chain and cause the majority of public health problems [34,36].
One of the hallmark findings of this study is the identification several virulence genes in the P.
aeruginosa isolates that might contribute to the pathogenicity of this bacterium in multiple hosts.The presence of the algD and lasB genes in around 32.0% and 9.0% of the P. aeruginosa isolates, respectively, suggests the potential virulence of these strains.The algD gene is associated with alginate production, which contributes to the formation of biofilms and enhances the bacteria's resistance to host immune responses and antibiotics [49,50].The lasB gene encodes for elastase, an enzyme involved in tissue damage and immune evasion [51].On the other hand, the low prevalence of the exoA gene (4.55%) and the absence of the exoS gene indicate the variability in the distribution of virulence factors among these isolates.The exoA gene encodes exotoxin A, a cytotoxin known to interfere with host cell function, contributing to cytotoxicity and the establishment of infections [52].Therefore, the differential presence and absence of these virulence genes among the isolates underscore the heterogeneity of P.
aeruginosa strains and their potential variation in virulence and pathogenicity.

Conclusions
To the best of our knowledge this is the first report on detection of MDR P. aeruginosa from various environmental samples including non-hospital residential environment (BAURA), hospital-based clinical (human and animal hospitals) and poultry markets samples for the first time in Bangladesh.We found that P. aeruginosa is prevalent in these diverse group of samples at 20.0%, and highest prevalence was recorded in poultry samples (30.0%).Despite having discrepancy in prevalence according to sample types, 100.0%P. aeruginosa isolates showed resistance against at least five antibiotics tested.Additionally, identified MDR P. aeruginosa were found to harbor beta-lactamase genes such as blaTEM, blaCMY, blaSHV, and blaOXA and virulence genes including exoA, lasB, and algD.Occurrence of MDR in P. aeruginosa is very alarming, since human and other animals can easily pickup these potential pathogens from the environment to get infection.Routine surveillance and practice of regular disinfection of environmental surfaces could be adopted rigorously to reduce their load in the environments.
Indeed, there are several promising avenues for further research on the P. aeruginosa isolates obtained in this study.Conducting further investigations using advanced techniques such as whole-genome-based phylogenetic analysis, epidemiological source tracking, sequence typing of isolated P. aeruginosa strains, and determining the minimum inhibitory concentration (MIC) of antibiotics, resistome, and pathogenicity profiles would significantly contribute to a comprehensive understanding of various aspects related to P. aeruginosa infections.
Leveraging a larger cohort of samples in these investigations would increase the robustness and representativeness of the findings, leading to more reliable conclusions and facilitating the development of more effective strategies for infection control, treatment, and management of P. aeruginosa infections.Each subscript letter denotes a subset of genes categories whose column proportions do not differ significantly from each other at the 0.05 level.NA: Not applicable.

Table 1 .
Primer sequences used in this study.

Table 4 .
Association between phenotypic and genotypic resistance patterns of isolated P.