Introduction

Pseudomonas aeruginosa, an opportunistic pathogen, has been associated with broad spectrum of infections such as pneumonia, urinary tract infections, and bacteraemia. It is the leading cause of life threatening nosocomial infections among the patients with impaired immune systems, such as neutropenia or cancer. Selection of appropriate antibiotics in pseudomonas infections pose a therapeutic challenge as they develop (adaptive) resistance during the course of treatment in addition to inherent and acquired resistance to different antibiotics (Bajpai et al., 2019).

Antimicrobial resistance (AMR) in Pseudomonas aeruginosa involves several mechanisms, such as induction of beta-lactamases, membrane permeability alterations and extrusion by efflux pumps. Acquired resistance involve mutation or the acquisition of gene encoding resistance determinants. It includes plasmid mediated betalactamases such as Amp C, Extended spectrum betalactamase and MBL (Abbas, El-Ganiny, & Kamel, 2018). The spread of extended-spectrum betalactamase (ESBL)-producing clinical isolates of P. aeruginosa has dramatically increased worldwide and frequently reported in India. This “evolving crisis” is regarded as public health threat as it limits the therapeutic choices. ESBLs are a group of plasmid mediated β-lactamases that hydrolyse penicillins, extended spectrum cephalosporins (3^rdgeneration) and monobactams (aztreonam). However, they are inhibited by cephamycin, β-lactamase inhibitors, such as clavulanic acid and carbapenem (Pragasam, Veeraraghavan, Nalini, Anandan, & Kaye, 2018). According to Ambler classification, ESBL-type enzymes are categorized into classes A and D. First ESBL identified in P. aeruginosa was PER-1 (Pseudomonas extended spectrum betalactamase) which share only 25% to 27% homology with TEM and SHV -type ESBLs. Other Class A ESBL type enzymes frequently reported in P. aeruginosa are GES, VEB, BEL and PME in contrast to the dominance of CTX-M, SHV, and TEM ESBL in Enterobacteriaceae. However, in a few isolates, classical type A enzymes such as TEM-4, TEM-21, TEM-24, TEM-42, SHV-2a and SHV-12 was described. Class D - oxacillinase that (OXA-2 and OXA-10 derivatives, OXA -4 and OXA-18) have extended substrate profiles have been reported in P. aeruginosa. The most common ESBLs reported in P. aeruginosa are VEB, OXA, and PER types (Laudy et al., 2017). ESBL producing Pseudomonas aeruginosa due to different types of enzymes, with wide range of substrate profile, emphasizes the need for detection of such isolates and their antimicrobial susceptibility pattern. This would prevent not only the therapeutic failures but also reduce the length of stay in hospital and nosocomial outbreaks. Hence, the present study was undertaken

• To Identify and isolate Pseudomonas aeruginosa from clinical samples

• To identify the prevalence of ESBL producing Pseudomonas aeruginosa among the clinical isolates.

• To determine the antimicrobial resistance profile of ESBL producing Pseudomonas aeruginosa

Materials and Methods

This study is a prospective study conducted at Clinical microbiology Laboratory of Sri Muthukumaran Medical college hospital and research Institute for a period of 9 months. Institutional ethical committee approval was obtained. All samples such as pus, urine, blood, sputum, ear swab, wound swab and different body fluids received for bacterial isolation from ICUs, OPD and IPD during the study period were analyzed. Blood agar and Macconkey agar plates were inoculated with sample and incubated at 37C for 24-48hrs. Provisional identification was done by Gram staining morphology, colony characteristics, motility, Catalase and Oxidase test. Pseudomonas aeruginosa was confirmed by standard microbiological procedures (Tille, 2014). Samples showing growth positivity to Pseudomonas aeruginosa was included in the study.

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing of the isolates was done by Kirby-Bauer disc diffusion method on Mueller-Hinton agar according to CLSI guidelines. Following antipseudomonal antibiotic discs were used, gentamicin (10μg), amikacin (30μg), ciprofloxacin (5μg), Levofloxacin (5μg), Ceftazidime (30μg), Cefepime (30 μg), Ampicillin (10μg), Piperacillin (100 μg), Piperacillin-tazobactam (100μg/10μg), Aztreonam (30μg), and Imipenem (10μg). The zones of inhibition were interpreted as per CLSI guidelines (CLSI, 2019). Pseudomonas aeruginosa ATCC 27853 was used as the control organism for antibiotic sensitivity. MIC of polymyxin and colistin was determined using E test and the elliptical zone of inhibition intersecting with MIC strip was interpreted as per CLSI guidelines (CLSI, 2019).

Phenotypic Confirmatory Disc Diffusion Test (PCDDT)

Isolates with intermediate or resistant susceptibilities for extended spectrum cephalosporin, Ceftazidime or aztreonam were considered as probable ESBL producers.ESBL production is confirmed by Phenotypic Confirmatory Disc Diffusion Test. Lawn culture of a test isolate adjusted to 0.5 Mc farland suspension was made on MH agar. A ceftazidime (30 μg) and ceftazidime / clavulanic acid (30 μg/ 10 μg) discs were placed aseptically on the MH agar plate at a distance of about 20 mm from edge to edge and the cultures were incubated at 37°C overnight. The observation of a ≥ 5mm increase in the zone diameter for ceftazidime with clavulanic acid compared to ceftazidime alone was considered positive (CLSI, 2019). (Figure 1)

Results

During the study period 312 bacterial isolates were obtained. Out of 312 isolates,69 non duplicate isolates of Pseudomonas aeruginosa obtained from patients attending our tertiary care hospital were analyzed. Rate of occurrence of Pseudomonas aeruginosa is 22.11% (69/312). All isolates were screened for ESBL production based on routine antimicrobial susceptibility testing results. 36 Isolates with intermediate or resistant susceptibilities for extended spectrum cephalosporin, Ceftazidime or aztreonam were considered as probable ESBL producers and subjected for phenotypic confirmatory disc diffusion test (PCDDT).

Sixteen (16) isolates were confirmed as ESBL producers by PCDDT. Hence, prevalence of ESBL producing pseudomonas aeruginosa(16/69) in this study was 23.18%. (Figure 2)

Pseudomonas aeruginosa isolates were obtained from patients with skin and skin structure infections (29/69), urinary tract infections pneumonia (16/69), Ear infections (8/69), bloodstream infections (8/69) and other infection types (16/69). Maximum ESBL isolation rate was obtained from pus 6(37.5%) followed by Urine 3(18.75%) and Ear swab 3(18.75%) as shown in the Figure 3.

81.25% of ESBL isolates were from in patient department while 18.75% from outpatient department of our hospital.

Out of 16 isolates maximum isolation rate was from medicine department (31%), followed by 18.75% from ENT,12.5% from surgery and 12.5% from ICU and 6% from OBG (Figure 4).

In our study, agewise distribution of ESBL isolates of Pseudomonas aeruginosa were common in the age group of 41-60 yrs(56.25%) followed by 21-40 yrs (18.75%)and 0-20 yrs(18.75%). (Figure 5). Among the 16 patients from whom ESBL was isolated,75% (12) of them were Males and 25% were females(4). Male to Female ratio was 3:1.

Multi drug resistance P. aeruginosa (MDRPA) is defined as an isolate intermediate or resistant to one agent in at least three antimicrobial classes (β- lactams, carbapenems, aminoglycosides, and Fluoroquinolones). 34 isolates were observed to be Multi Drug Resistant (MDR). Hence, the isolation rate of MDRPA was 49.27%. Prevalence rate of ESBL isolates among the MDR strains was 41.17%(14/34).2 isolates which were ESBL positive was observed to be Non MDR (Table 1). We observed extremely significant association between MDR isolates and ESBL production (p value =0.0005).

Out of 69 isolates,16 were ESBL producer and 53 were non ESBL producers. Resistance profile to antipseudomonal antibiotics by ESBL versus non ESBL isolates as follows, Penicillins (Ampicillin 62.5% versus 40%, Piperacillin 62.5% versus 32%), Aminoglycosides (Amikacin 44% versus 17%, Gentamicin 75% versus 70%), Fluoroquinolones (Ciprofloxacin 63% versus 34%, Levoloxacin 38% versus 32%), Cephalosporins (Ceftazidime 100% versus 32%, Cefepime 25% versus 17%), monobactams (Aztreonam 50% versus 23%) and betalactam- betalactamase inhibitor (Piperacillin Tazobactam 37.5% versus 15%) and Carbapenem (Imipenem 12.5% versus 87.5%). All isolates were sensitive to Polymyxin and Colistin. The results show that ESBL production not only effect sensitivity to β- Lactam antibiotics but these strains are simultaneously resistant to many other classes of antimicrobials which are commonly used as empirical treatment in Pseudomonas infection. We found statistically significant difference (p< 0.05) in resistance among ESBL producing and non-ESBL strains in Ceftazidime and Piperacillin. (Figure 6). 33.3% of ear swab and 100% of blood and bodyfluid isolates were resistant to imipenem. (Table 2)

Discussion

Pseudomonas aeruginosa is well known for its ability to utilize several antimicrobial resistance mechanisms. It includes production of ESBL, loss of outer membrane protein, production of class AmpC beta-lactamases, active efflux pump and altered target sites. Foremost among the mechanisms of resistance is the production of Extended-spectrum β- lactamases. Often, genes for this enzyme is carried on plasmids, facilitating rapid spread between microorganisms. Emergence of β -lactamase producing Pseudomonas aeruginosa presents major diagnostic and therapeutic challenge in the management of infection.

In our study, PCDDT confirmed 23.18% of clinical isolates were ESBL producers. Our results are comparable to the recent studies (Das, Azad, Bimal, & Oraon, 2020; Jeyabharathi & Rejitha, 2018) and (Shaikh, Fatima, Shakil, Rizvi, & Kamal, 2015) who had reported prevalence rate of 28%, 26.5% and 25.13%. Even, similar study conducted in China reported 21% (Chen et al., 2015). However, studies from Uttarakhand and Central India (Ashish, Shailesh, & Ji, 2020; Bajpai, Pandey, Varma, & Bhatambare, 2014), reported high prevalence rate of 42% and 48%. These variations could be due to the fact that prevalence in any hospital settings depend on factors such as carriage rate among the hospital personnel, type of disinfectant used and antibiotic policy. This explains the need for surveillance for antibiotic resistance as a routine practice.

Maximum number of ESBL isolates were obtained from pus (37.5%) followed by Urine (18.75%). This was in accordance with the findings by similar studies (Jeyabharathi et al., 2018; Pramodhini & Umadevi, 2015) which reported 59.6% and 60% of isolates from pus respectively. In contrast to this observation high isolation rate from urine specimens was reported (Nithyalakshmi, Vidhyarani, Mohanakrishnan, & Sumathi, 2016). This observation is of great concern as the study was conducted previously in the same institute. Hence, ESBL associated type of infections not only varies from place to place even in the same place over a period of time.

Majority (81.25%) of ESBL isolates were from in patient department with maximum isolation rate from medicine department (31%) while 18.75% from outpatient department of our hospital. This correlates well with the previous literatures (Anupurba, Battacharjee, Garg, & Ranjansen, 2006; Ashish et al., 2020). It may be due to exposure to multiple antibiotics in a hospitalized patients leading to selective pressure for the emergence of resistant strains.

Many studies have recognized the important role of age in patient’s susceptibility to pseudomonas infection. In our study, rate of isolation of ESBL producing Pseudomonas aeruginosa from persons aged 41-60 yrs was 56.25% followed by 21-40 yrs (18.75%)and 0-20 yrs(18.75%). This is in corroboration with the findings by the Kothariashish et al, who showed that high rate of isolation was in the age group of 46 -60yrs. He expressed his view that this could be attributed to the facts that this age group (46-60) goes out of home and were at utmost risk to acquire an infection (Ashish et al., 2020; Mullai, Bhuvaneshwari, Jagadeeshwari, Aruna, & Kalyani, 2019) Male preponderance (75%) was observed in this study which is comparable to the findings which reported 66% by a similar study (Khan, Iqbal, Rahman, Farzana, & Khan, 2008). However, analysis of 18.75% of ESBL isolates from urine sample showed 66.6% from female patients. This was in harmony with the fact UTI caused by Pseudomonas aeruginosa is more common in females.

Increasing incidence of Multidrug resistant Pseudomonas aeruginosa (MDRPA) is alarming as they are difficult and expensive to treat. In our study, rate of isolation of MDRPA was 49.27%. Our findings was supported by researchers (Biswal, Arora, Kasana, & Neetushree, 2014; Prakash, 2014; Sabetha, Nithyalakshmi, & Nirupa, 2017) who had reported 35.18%, 36.12% and 31.73% respectively. In a similar studies high rate of 85.54% and 75.8% of MDRPA isolates was also documented (Dawra, Sharma, Bachhiwal, & Vyas, 2017; El-Baky, El-Azeim, & Gad, 2013). Capsoni et al in his recent study reported MDR isolates should be considered as a significant independent risk factor for mortality. This clearly indicates variable incidence rate of MDRPA and the need for judicious use of empirical drugs in Pseudomanas infections (Capsoni, Bellone, & Aliberti, 2019).

Majority of MDR isolates were ESBL producers(41.17%). Association between ESBL and MDR has great impact on mortality which suggests the ESBL coverage when empirical drug is initiated in patients with Multidrug resistant bacteria to reduce the mortality.

ESBL isolates exhibited more resistance against antipseudomonal antibiotics tested compared to non ESBL. In this study, all ESBL isolates were sensitive to Polymyxin and Colistin such in vitro activity was reported by previous literature (Gupta et al., 2006; Kaur & Singh, 2018)

88% of ESBL isolates were Imipenem sensitive. Carbapenem resistance may be due to coexistence of MBL genes along with ESBL which confers resistance to Imipenem or altered outer membrane permeability or active efflux pump. Though the results suggest them to be good choice of drug, in a healthcare setting, development of resistance might be due to heavy selective pressure. Hence, we strongly suggest that antibiotic policy must reserve this drug and used only in life threatening infections.

In our study, Piperacillin-Tazobactam (beta lactam and betalactamase inhibitor combination) observed to have low resistance profile (37.5%) compared to piperacillin alone (62.5%). This was in harmony with 36% susceptibility to Piperacillin -tazobactam reported (Kadry, Serry, El-Ganiny, & El-Baz, 2017). Our study highlights that piperacillin-tazobactam are good choice for empirical therapy.

Although belonging to cephalosporin group, we observed variable results with reference to generations. All ESBL isolates were resistant to Ceftazidime, but in vitro activity of Cefepime was relatively lesser (25%). Production of ESBL by the isolate involved might contribute to this high resistance.

Among the aminoglycosides, Amikacin showed the least resistance of 44% which suggests that it can be recommended compared to gentamicin (75%). Our results were similar to the findings by previous studies (Kaur et al., 2018) and (Shaikh et al., 2015) who reported among ESBL isolates, 39.1% were amikacin resistant and 94.74% were gentamicin resistant.

Ciprofloxacin and Levofloxacin resistance was observed to be 63% and 38% respectively which are in agreement with the findings 66.7% and 59.8%, reported by Kaur et al. As Fluorouinolones are concentration dependent antibiotic, routine use of ciprofloxacin may be the reason behind this difference among the same group. (Kaur et al., 2018).

Co-resistance to antibiotics among ESBL isolates have been considered as a serious burden even at our centre. Occurrence of genes encoding resistance to aminoglycoside, and quinolones on the same plasmid that encodes for ESBL production may contribute to this co-resistance.

Analysis of resistance profile of ESBL isolates according to the samples revealed imipenem resistance isolates in ear swab, blood and bodyfluids. Imipenem resistant ESBL pseudomonas aeruginosa has been frequently isolated from blood and respiratory samples. Emergence of such resistance in different types of sample suggest the need for their antimicrobial susceptibility surveillance to prevent further dissemination.

Conclusion

We demonstrated ESBL isolates with high resistance rate than Non ESBL, reiterating the need to identify them as a routine in a laboratory to initiate appropriate antibiotic therapy. Our study findings would be useful to formulate our antibiotic policy so that it could optimize the utilization of armamentarium of antibiotics in an effective way.