Antimicrobial resistance pattern of Uro-Pathogens emphasizing non-lactose fermenting gram negative bacilli

Objective: To identify various species of non-lactose fermenting gram-negative bacilli involved in urinary tract infections, and to determine their antimicrobial resistance pattern. Method: The retrospective, descriptive, cross-sectional study was conducted from January 1 to April 1, 2022, at the Dow University of Health Sciences, Karachi, and comprised data from the institutional diagnostic laboratory that was related to urine samples regardless of age and gender from January 1, 2020, to December 31, 2021. Data was analysed using SPSS version 25. Results: Of the 103,887 urine samples, 41,280(39.7%) were positive, 51,146(49.2%) showed no bacterial growth, 11,000(10.6%) had non-significant bacterial growth and 461(0.4%) had mixed bacterial growth. Of the positive samples, 18359(44.5%) were positive in 2020, and 22,921(55.5%) in 2021. Gram-negative lactose fermenting bacteria included escherichia coli 23,123(22.3%) and klebsiella pneumoniae 2,993(2.9%), gram-negative non-lactose fermenting bacteria included pseudomonas aeruginosa 1,110(1.07%), and gram-positive bacteria included enterococcus 8,008(7.7%). Pseudomonas aeruginosa was most resistant against tobramycin 880(79.3%) and least resistant against piperacillin-tazobactam 146(13%). Conclusion: Piperacillin-tazobactam was highly sensitive drug against non-lactose fermenting uro-pathogens.


Introduction
Gram-negative bacilli are common pathogens associated with urinary tract infections (UTIs).These gram-negative bacilli are becoming more resistant to several antibiotics with time. 1 The gram-negative organisms that cause frequent onset of UTIs are mainly escherichia (E.) coli and klebsiella (K.) pneumonia. 2UTI is among the most common bacterial infections affecting all age groups.UTI is one of the major public health concerns in terms of illness and financial burden, contributing greatly to healthcare expense.It has been recently estimated in the United States that around 8.6 million healthcare visits were made annually, resulting in serious global economic burden 3 .Prevalence of UTI is affected by multiple factors, such as old age, poor personal hygiene, pregnancy, urinary catheterisation, genitourinary tract abnormalities, and co-morbidities, including diabetes and human immunodeficiency virus (HIV) infections.Clinically, UTI may present as asymptomatic, acute, chronic, complicated, or uncomplicated, depending upon the type of infectious agent, part of the urinary tract involved, and patient's immune response. 4Most UTIs are treated empirically based on the culture and sensitivity pattern generated by microbiology laboratories.The spectrum of antimicrobial susceptibility of UTI pathogens varies from time to time and in different geographic areas.Therefore, regular monitoring of the antibiotic susceptibility pattern of these uro-pathogens is required for reduction and eradication of antimicrobial resistance.The antimicrobial resistance against broad-spectrum antibiotics is increasing with every passing day owing to excessive and irrational utilisation, self-medication, mal-drug disposal practices, and genetic proliferation among bacterial species. 5Is are more common in females than males due to anatomical proximity of urethra to the gut opening. 6UTI signifies common illnesses confronted in medical practice today, with an expected 150 million UTIs each annum globally.Around 20% of UTIs are documented to be found in males.In healthy women, uncomplicated UTIs have a prevalence of 50/1000/time. 7Besides, 50% of females experience UTI incidence at least once in their lifetime, and 20-40% females have chronic episodes. 8ost incidents of UTI are triggered by E. coli, while K. pneumoniae, staphylococcus (S.) saprophyticus and proteus species represent the remaining infections. 2 Susceptibility data from regional microbiological services help in the empirical range of antibiotics for treatment of UTI, though such data is restricted to complex UTIs as uncomplicated UTI samples are hardly sent to the laboratories.The growing prevalence of drug resistance amongst uro-pathogens is considerable for public health interest, requiring continuous antibiotic susceptibility screening for bacteria causing UTI.Additionally, antimicrobial sensitivity for UTI-causing bacteria is documented to change with time and location.Thus, screening for sensitivity is significant for creating up-todate epidemiological information. 9The heterogeneous group of proteobacteria, non-lactose-fermenting gramnegative bacteria (NLF-GNB) is distinguished by the failure to ferment carbohydrates for energy production to carry out their essential cellular purposes.In UTIs, NLF-GNBs must not be overlooked as they are essential bacteria from the epidemiological perspective with greater prevalence. 10These contain, in descending order of occurrence, pseudomonas, acinetobacter, Burkholderia cepacia complex (BCC), and stenotrophomonas (xanthomonas) maltophilia.Additionally, there are achromobacter, alcaligenes, brevimundas, elisabethkingia, flavobacterium, ralstonia and others.Some less predominant members, for instance Burkholderia (B.) mallei and B. pseudomallei, acquire significance as bioterrorism representatives. 11They are very common in cunning infections, impacting harshly immunocompromised and weakened patients aged around 60 years.
The current study was planned to identify various species of NLF-GNB involved in UTIs, and to determine their antimicrobial resistance pattern.

Materials and Methods
The retrospective, descriptive, cross-sectional study was conducted from January 1 to April 1, 2022, at the Dow University of Health Sciences (DUHS), Karachi, and comprised data from the institutional diagnostic laboratory that was related to urine samples regardless of age and gender from January 1, 2020, to December 31, 2021.
Duplicate samples, catheter specimens, specimens with growth of candida species, data of antibiotic susceptibility patterns of all isolated gram-positive bacteria and lactosefermenting gram-negative bacteria of other sources, like blood, pus, pleural fluids, etc., were excluded.

Discussion
UTI is the most prevalent infection affecting people worldwide, and is rated as the third most common infectious disease.Uncomplicated UTIs are cured with short courses of antibiotics, but urine samples are sent in several cases for microbiological assessment following treatment failure or chronic or worsening infection. 14The most consistently associated infectious agents of UTIs are enterobacterales, including E. coli and klebsiella, citrobacter, enterobacter, serratia, proteus, morganella and providencia species, gram-positive cocci, including enterococcus species, S. aureus and S. saprophyticus, and NLF-GNBs, like P. aeruginosa. 11e current study had 41,280 urine samples positive for bacterial growth.Females of reproductive age formed the primary group of patients with UTI, which was in line with literature that has extensively reported that women have a greater occurrence of UTIs than males, primarily due to anatomic and physical issues. 15Similarly, a study documented that elderly woman aged >61 showed lower UTI incidence (22%) that males.This was perhaps because with age, males develop prostate swelling and neurogenic bladder. 16 the present study, among gram-negative uropathogens, E. coli had the highest prevalence, followed by K. pneumonia, which has been reported earlier as well. 10,17The enterococci species was the dominant pathogen compared to other gram-positive isolates, having a slightly higher prevalence than a study in 2016. 18NLF-GNBs are frequently correlated with uropathogenecity. 19Among NLF-GNBs, most of the urine infections were caused by P. aeruginosa and species, which is similar to the data reported earlier. 20These two isolates showed increased resistance pattern against AMK, TZP, CAZ and CIP.However P. aeruginosa was found to be more resistant towards MEM and TOB,.Acinetoobacter isolates wereresistant against CRO.The findings closely resembled those reported earlier. 21However, contradictory findings have also been reported. 22In the present study, proteus mirabilis and proteus vulgaris showed resistance against several antibiotics, but higher resistance was observed with CFM and CRO.This was in line with another study. 23-GNBs represent a challenge to clinicians because of their rising resistance against various antibiotics classes, ultimately leading to multidrug resistance (MDR), extensive drug-resistance (XDR), or even pan-drug resistance (PDR). 24The resistance in these clinical isolates has developed owing to various mechanisms, such as intrinsic non-susceptibility processes, alterations or through plasmids/integrons during the extended course of therapy although the same treatment was effective primarily.The resistance mechanism contains porin deficiency and mutations impacting outer membrane absorbency (β-lactam antibiotics), changes in target locations (aminoglycosides, fluoroquinolones), energyreliant efflux pumps (fluoroquinolones), as well as the making of drug-inactivating enzymes. 25e current study has limitations as it did not deal with UTI prevalence in the local population.Besides, the current data could not correlate antimicrobial resistance with the duration and cause of UTI because it was retrieved from the laboratory records in which clinical data is not recorded.

Conclusion
Among NLF-GNBs, P. aeruginosa and acinobacter species were the most frequent cause of uro-pathogenecity, with great resistance towards MEM, TOB, AMK, TZP, CAZ and CIP.The There is an urgent need for antibiotic stewardship and infection control measures in healthcare settings.

Figure :
Figure:Frequency of various species of non-lactose fermenting gram-negative uro-pathogens.
MacConkey agar was later identified by colony's morphology, gram staining and various biochemical tests, including catalase test, oxidase reaction, citrate test, sulfur indole motility (SIM) test, triple sugar iron (TSI) test and urease test, while the final confirmation was done by Analytical Profile Index ()API 20E and API 20NE (bioMerieux, France), as needed.Antibiotic susceptibility test was done using the Kirby-Bauer disk diffusion test protocol with 0.5 McFarland turbidity standard.

Table - 1
: Mean organ weights (g) and ventricle thicknesses (cm) of the study population.