European Journal of Cardiovascular Medicine

Pseudomonas species are a group of gram-negative, aerobic, oxidase positive, pigment producing, non-fermenting non-spore bearing bacilli. It is an opportunistic pathogen, exploiting some break in the host defenses to initiate an infection. Its ability to grow in a wide range of temperatures (5-45°C), virulence factors like pilli, alginate coat, pigment, and toxin production helps the organism to evade the host immunity and cause tissue injury. (1,2)

As opportunistic pathogens, they can cause various infections in immunocompromised hosts like patients with cystic fibrosis, neutropenia, burns, cancer, AIDS, organ transplant, uncontrolled diabetes mellitus, as well as immunocompetent hosts. (1) The mortality of nosocomial pseudomonal pneumonia is approximately 70% in immunocompromised hosts. Pseudomonas species are ubiquitous microorganisms that can survive on minimal nutritional requirements and can persist in varied environmental surroundings. These properties help the organisms to survive in both community and hospital settings. In the hospital, they can be isolated from a variety of sources, including ventilators, endoscope washers, antiseptics, tap water, sinks, mops, injectables, etc. (2,3) In the community, their reservoirs include, vegetables, water bodies, contact lens solutions, home humidifiers, etc. (4,5,6)Pseudomonas aeruginosa can cause various infections like folliculitis, burn wound infections, osteomyelitis, pneumonia, otitis externa, etc, and nosocomial infections like ventilator-associated pneumonia, catheter-associated urinary tract infections, etc. (7,8)Along with its ability to survive in harsh environments, P.aeruginosa displays multiple drug resistance mechanisms which include intrinsic resistance due to the presence of efflux pumps and decreased outer membrane permeability and plasmid mediated beta-lactamase enzymes (ESBL, AmpC, and carbapenemases)production along with the ability to form biofilm. (7,9) P. aeruginosa is one of the MDR ESKAPE pathogens, which stands for pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter. (10,11)

The following retrospective study was undertaken to find out the prevalence and trend of antibiotic resistance patterns of Pseudomonas species with special emphasis on Pseudomonas aeruginosa in all clinical samples in a tertiary care hospital in Eastern India.

The present study was undertaken in Medical College & Hospital, Kolkata, India after obtaining the Institute Ethics Committee approval, from April 2021 to March 2023. A record based retrospective analysis of data of all clinical samples of all the indoor patients admitted in the tertiary care center received over the 2 years yielding growth of Pseudomonas species was taken.

The various biological samples like urine, pus, sputum, ET aspirate, blood, bronchoalveolar lavage, ascitic fluid, cerebrospinal fluid (CSF), etc of the admitted patients were sent to the microbiology laboratory for testing. The culture was done on MacConkey’s medium and Nutrient agar. Identification of the bacterial isolates was done based on standard recommended procedures. The strains were identified based on the colony morphology and gram staining. Further processing was done either by conventional methods or by automatic method (Vitek 2 Compact) whichever was available as per CSLI guidelines 2021. Conventional methods included strain identification by oxidase test, pyocyanin pigment production, ability to reduce nitrate, use of citrate and malonate as carbon sources, and growth at 42°C. Control used was P. aeruginosa ATCC 27853. Antibiotic susceptibility for P. aeruginosa was performed using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar as per CSLI guidelines using commercially available disks (Hi-Media): Amikacin (30 μg), Gentamycin (10µg), Ciprofloxacin (5μg), Levofloxacin(5µg), Ceftazidime (30 μg), Ampicillin(10μ), Piperacillin (100 μg), Cefepime (30 μg), Cotrimoxazole(25µg), Imipenem (10 μg), Meropenem (10 μg), and Piperacillin-Tazobactam (100/10 μg). The results based on the zone size, as compared with the standard strains, were interpreted as Sensitive or Resistant as per the recommendations of the CLSI manual. Automated method (Vitek 2 Compact) was also used for identification and Antibiotic susceptibility testing as per CSLI guidelines as per availability. (2,12) Since it was an observational retrospective study and did not possess any intervention, hence the consent part was waived. All the data were analyzed in Microsoft Excel 2010 and GraphPad Prism software.

A total of 206 Pseudomonas isolates from various clinical samples comprised 116 (56.3%) males and 90 (43.7%) females, with a male: female ratio of 1.28:1. The age of the patients ranged between1 month and 80 years, with a median of 40 years. Age-wise and sex-wise distribution of Pseudomonas isolates is shown in Table 1. Clinical sample wise distribution of all Pseudomonas isolates is shown in Table 2. The most common isolate of Pseudomonas spp was Pseudomonas aeruginosa (143 samples; 69.2%) from all clinical samples. Other isolates were P.stutzeri (5; 2.5%),P.putida (4; 1.9%), P.luteola (3; 1.5%) and other Pseudomonas spp (51; 24.8%). Out of the 143 isolates of P. aeruginosa, 64 (44.75%) were from pus and wound swabs, 28 (19.58%) were from blood, 17 (11.89%) were from ET aspirate, 13 (9.09%) were from sputum, 13 (9.09%) were from urine, and 8(5.59%) were from other samples like BAL fluid (5 samples), Bile aspirate (2 sample) and Implant (1sample).The distribution of P. aeruginosa among various clinical samples is shown in Fig. 1. The Sensitivity pattern of all Pseudomonas species is shown in Table 3. The antimicrobial resistance pattern of P. aeruginosa isolates is shown in Fig 2. Resistance to ceftazidime, and cefepime were 43.4%, and 49.6% respectively. Resistance to fluoroquinolones like ciprofloxacin and levofloxacin was 32.8% and 35% respectively. Aminoglycosides, amikacin and gentamycin showed a resistance of 34% and 36% respectively. Imipenem and meropenem showed 37% and 35% resistance respectively. Resistance to piperacillin was 43.4% while piperacillin-tazobactam was 35%. Resistance to cotrimoxazole was 37.7%. Some isolates (4.9%) also showed resistance to polymyxin and colistin.

Table 1: Age‑wise and sex‑wise distribution of Pseudomonas isolates:

Table 2: Clinical sample wise distribution of all Pseudomonas isolates:

Table 3: Sensitivity pattern of all Pseudomonas species:

Fig 2: Percentage of resistance among P.aeruginosa isolates (n=143)

Pseudomonal infections may be due to both exogenous and endogenous sources. They account for a significant number of morbidities and mortalities particularly among immunocompromised and debilitated patients. (8) A total of 206 isolates of Pseudomonas species were isolated among which Pseudomonas aeruginosa was isolated among 143 clinical samples. Sex-wise distribution among males and females was 1.28:1 similar to Dash et al and Kanthkumar et al studies which reported male female ratio as 1.4:1 and 1.1:1 respectively. (13,16)  The predominant age group affected was between 19 – 60 years (63.6%) followed by the elder age group >60 years (19.9%) which is similar to studies by Kanthkumar et al and Chander et al. (16,17) Highest number of Pseudomonas isolates were from Pus samples (44.75 %) in our study which was in concordance with several studies conducted in different regions of India, however, Javiya VA et al reported maximum isolates of Pseudomonas from urine samples. (16-18,15) 

 An alarming high resistance of P.aeruginosa to Penicillin and Cephalosporin group has been accounted for in studies by Javiya VA et al and Kanthkumar et al, similar to our study (about 43.4% and 49.6% respectively), and are now hardly considered as an option to treat nosocomial Pseudomonal infections. (15,16) Kanthkumar et al had also reported 33% resistance of Pseudomonas to fluoroquinolones in a study conducted in 2022. (16) Studies by Gasink et al showed that there has been a significant rise in fluoroquinolone resistance among Pseudomonas isolates from 15% in the year 1991 to 41% in 2000 (19). Shahid M et al noted gentamycin resistance among these isolates of about 32.6% similar to that of our study. (20) Genes conferring resistance to carbapenems is a matter of much

concern not only among P.aeruginosa but also species like P.putida, P.stuzeri, etc. (14,21) With the increase in multi drug resistance patterns in Pseudomonas aeruginosa isolates over the years, particularly the nosocomial strains, selection of appropriate treatment strategy becomes very difficult. Appropriate antibiotic therapy must be accompanied by effective infection curtailing measures such as removal of infection sources like infected Foley’s catheter, IV-line, ET tube, and wound debridement procedures. (14)

Pseudomonas aeruginosa infections are difficult to treat as they have limited susceptibility to anti-microbials and have an inclination to develop further resistance due to horizontal gene transfer. A knowledge of these infective agents and determining the antimicrobial susceptibility pattern in a particular geographical setting is essential. Such studies would help in formulating the local antimicrobial guidelines, curtail economic burden, decrease morbidity and mortality, and increase the quality of life of patients.

Source of Support: Nil

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