Phenotypic and genotypic characterization of multidrug-resistant isolates from patients with catheter-associated urinary tract infection in a tertiary care hospital

Introduction

Urinary tract infections (UTIs) are the most common infections occurring in people. In critically ill patients, catheter-associated (CA) UTIs can lead to bacteremia, and they are the leading cause of morbidity and mortality in approximately 10% of hospitalized patients.[1] CA-UTI accounts for over 80% of infections in catheterized patients admitted in intensive care units (ICUs) during their hospital stay.[2] The important predisposing factors commonly associated with CAUTIs are conditions such as diabetes, immunosuppression, renal insufficiency, and urinary incontinence commonly among neurologic and orthopedic patients.[12] Other factors for CAUTI could be patients staying on catheter for a prolonged duration of time, female and elderly patients, patients with severe illness, catheterization performed under nonsterile conditions, and catheter insertion by undertrained professionals.[34] The important factor that help to reduce CAUTIs is catheterizing the patient only when absolutely necessary and by shortening the duration time of indwelling catheters. The incidence of multidrug resistance in organisms is increasing due to dissemination of resistance determinant genes mediated by transposons, plasmids, and gene cassettes in integrons.[3] Beta-lactam NDM-1 is a novel metallo-beta-lactamase with divalent Zn ions at its active site and has the ability to confer resistance to almost all the β-lactams. The NDM-1 gene is normally carried on a variety of plasmids along with other resistance factors. The bacteria also show broad antibiotic resistance to all classes except colistin. OXA-23 may be chromosomally and plasmid mediated and encodes for serine carbapenemases widely present among Acinetobacter species and is responsible for its extreme drug resistance. Very rarely, OXA-23 group is seen in Enterobacteriaceae. Resistance to carbapenems due to carbapenemase production poses serious challenges in the treatment of such infections with pan-resistant phenotypes.[5] Spreading of NDM-1 gene in a hospital may be a complex event involving several modes of spread.[6] This study aims to calculate the prevalence of CAUTI, the common pathogens involved, to phenotypically and genotypically characterize the multidrug-resistant isolates from CAUTI patients, and to determine the prevalence of NDM-1 and OXA-23 genes involved in carbapenem resistance.

Materials and Methods

Samples were collected after obtaining informed consent from the patients or their attendants. Catheterized urine sample was collected aseptically following clamping and using a sterile collection container. The urine was collected through the catheter port. After cleaning the catheter with 70% isopropyl alcohol, it was clamped. After 30 min with the help of a sterile syringe, 1–2 ml of urine was aspirated and collected in a sterile container. The clamp was removed. Adult patients who were catheterized belonging to both sexes and admitted as inpatients in various wards such as ICU, medical wards, surgical wards, obstetric and gynecology wards, and orthopedic wards were included in this study. Samples were collected only from patients in whom the catheter has been in situ for a minimum of 48 h duration. Patients with preexisting renal disease were excluded from the study.

The urine sample collected from catheter was transported to the clinical microbiology laboratory without delay, within 1–2 h of sample collection, as urine is an excellent culture medium and contaminating bacteria might multiply and reach significant numbers.

Gram's stain of a smear made from urine was performed, and the presence of pus cells along with Gram-negative bacilli, Gram-positive cocci, and Gram-positive budding yeast cells was noted. The clinical significance of growth in urine cultures was estimated by the colony count of the organism per milliliter of urine. The urine was cultured on cysteine–lactose–electrolyte-deficient agar. This plate was incubated at 37°C for 18–24 h, and colony morphology was observed and noted the next day. As indwelling catheter specimens are collected through the urethra, the urine is likely to be contaminated by normal urethral flora, and colony counts are necessary for interpretation of culture. Samples found to have mixed growth of organisms were rejected, and a repeat sample was collected. Samples having >10⁵ colony-forming units per milliliter were processed further to identify the pathogen. For Gram-positive cocci and yeast, counts of 10⁴–10⁵ CFU/ml were considered significant. In general, counts <10³ CFU/ml were considered as contamination. In Gram's stain, the presence of pus cells and bacteria in every oil immersion field/20 fields indicate infection. Ninety-five percent of all UTIs were caused by a single type of organism. The routine biochemical tests for organism identifications such as indole test, triple sugar iron test, citrate utilization test, urease production test, mannitol-motility medium, and phenyl deaminase test were performed.

Results

A total number of 231 urine samples were collected from catheterized patients over a period of 3 months between June and August 2018 out of which 133 (57.6%) were males and 98 were females (42.4%). Seventy percent of these samples were from the ICU, and the rest were from other wards such as medicine, surgery, obstetrics and gynecology, and orthopedics. Of these, 101 samples (43.7%) yielded growth while the rest showed no/insignificant bacteriuria. Table 1 shows the organisms isolated in culture. Klebsiella was the most common organism to be isolated. Tables 2 and 3 show the antibiotic susceptibility pattern of all the Gram-negative bacilli and Gram-positive cocci isolated from these samples.

A total of 38 Gram-negative bacilli were resistant to carbapenems, of which 29 (76%) were from the ICU, 6 (16%) were from surgical wards, and 3 (8%) were from the medical wards. Of the 38 carbapenem-resistant Gram-negative bacilli, 23 were MHT positive and 15 were negative. Table 4 gives the distribution of organisms which were MHT positive.

Table 5 shows the PCR analysis of NDM-1 gene, and Table 6 shows the PCR analysis of OXA-23 gene. Figures 1 and 2 show gel documentation of the same.

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Figure 1

Polymerase chain reaction of NDM-1 was positive in 1, 9, and 10 and the band was 214 bp

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Figure 2

Polymerase chain reaction of OXA-23 was positive in 9 and the band was more than 453 bp

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In this study, 101 organisms were isolated, of which 72 were Gram-negative bacilli. Out of this, 38 showed carbapenem resistance, in that 23 were positive for MHTs and 8 (21.05%) were positive for NDM-1 gene. Of the 38 organisms tested for OXA-23 gene, only 1 (2.6%) was positive for the OXA gene.

Discussion

Of 231 samples collected, 95 samples were culture positive, of which 72 isolates were Gram-negative bacilli. This shows that the overall incidence of infection is 41%. Age-wise distribution revealed that maximum incidence was seen in those above 60 years of age. Bagchi et al.[7] in their study period of 2 years followed up 220 patients admitted at various medical, surgical wards and ICUs with respect to clinical features, risk factors, microbiological profile of CAUTI, and antibiotic susceptibility and found that 64 cases developed CAUTI and the incidence rate was 29.09%. This incidence is lower than that of the present study. Kazi et al.[8] studied 1380 catheterized patients, out of which 34 developed CAUTI. He demonstrated that if infection control practices are in place and monitored meticulously, it is possible to keep the incidence rates within the benchmark set by the individual hospital. In the present study, samples collected from ICUs were 70.1% and wards were 29.9%. Urine samples collected from ICUs had increased incidence of Candida spp. while those from other wards had E. coli in high incidence. However, the predominant uropathogen isolated from samples in our study was Klebsiella species, and E. coli was the second most common CA uropathogen after Klebsiella. Among the most common pathogens isolated in the ICUs, Candida spp. and Klebsiella spp. are most frequently encountered. In another study by Hanumantha and Pilli,[9] the predominant uropathogen was Citrobacter species, which was not isolated in this study. In a study by Vinod et al.[10] in Vellore, E. coli followed by Klebsiella sp were the common isolates in CAUTI. Therefore, it is inferred that the predominant pathogen varies from region to region depending on the local microbiological environment. In this study, the antibiotic susceptibility test result for the various organisms shows that all the Klebsiella isolates, Morganella morganii, and Proteus species and 50% of E. coli, Acinetobacter species, and most of the Pseudomonas isolates were resistant to the usual first and second-line antibiotics recommended for UTI, such as beta-lactams, aminoglycosides, and fluoroquinolones. In addition, increased resistance was seen for carbapenems such as imipenem and meropenem which leaves the clinician with no choice except for polymyxins. Organisms that produce carbapenemase enzyme are multidrug resistant. Increasing resistance to carbapenems that are most often the last line of therapy is being noted in hospital. The resistance to carbapenems may be related to either the decreased membrane permeability with overexpression of beta-lactamase or to expression of carbapenemases. In a study conducted by Agarwal et al.[11] in a tertiary care hospital in Northern India, 9.4% of E. coli isolates were positive for carbapenemase production and 7.2% isolates showed the presence of NDM-1 gene. In a study conducted in North East India by Bora et al.,[12]5% of E.coli isolates harbored the NDM-1 gene. Brennan et al.[13] reported carbapenem-resistant Enterobacteriaceae (CRE) in 61% of isolates from urine cultures, and a urinary catheter was present in 48% of these patients. Several studies carried out in different parts of India also have yielded similar results. Table 7 gives a comparative analysis of such studies which clearly shows that there has been an increase in drug resistance and prevalence of NDM-1 gene. In this study, out of the 38 CRE, 8 isolates were positive for the NDM-1 gene. Of these 8 isolates, 4 were Klebsiella, 2 were E. coli, Acinetobacter and Morganella one each, and one sample was positive for OXA-23. New Delhi metallo-beta-lactamase (NDM)-mediated carbapenem resistance in Pseudomonas aeruginosa and Acinetobacter baumannii is a major concern. The presence of NDM and its variants were studied in P. aeruginosa and A. baumannii at a tertiary hospital in North India by Rahman et al.,[14] and it has been found that 29.23% P. aeruginosa and 18.8% A. baumannii isolates were resistant to carbapenems and all of them were blaNDM positive. The single isolate which was positive for OXA-23 was Acinetobacter species. According to Khurana et al.,[15] the carbapenem resistance was higher in North than in South India. Gram-negative Bacilli blaOXA-1 was the most common carbapenemase gene among the South India strains, while blaNDM-1 followed by OXA-1 were the two most prevalent carbapenemase genes in the North Indian isolates.

Conclusion

This study has shown that carbapenem-resistant isolates from all the CA urinary tract-infected patients were 52.77% and most of them were Klebsiella. About 21% of them harbored the NDM-1 gene while only 2% had the OXA-23 gene. As all these genes confer multidrug resistance or pandrug resistance, it is very difficult to treat and adds to the morbidity and mortality rates. OXA-23 group is very notorious in spreading drug resistance among the superbugs Acinetobacter spp. So far it is not common among other Gram-negative bacteria, but as it is also plasmid mediated, we can soon expect it to spread and cause pandrug resistance among other organisms which are commonly isolated. There has been an alarming increase in the spread of carbapenem resistance which makes treatment of the patients very difficult, as most of the catheterized patients are in the ICUs and have several other associated comorbidities. To reduce the incidence of CA UTIs, it will be better if hospital infection control practices are in place.