CHARACTERIZATION AND ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF NON LACTOSE FERMENTING GRAM-NEGATIVE BACTERIAL ISOLATES IN A TERTIARY CARE TEACHING HOSPITAL OF CENTRAL INDIA

Objectives : The non-lactose fermenting Gram-negative bacilli (NLF-GNB) are notorious pathogens reportedly acquiring multiple drug resistance alarmingly and emerging as a public health threat globally. This study was conducted to isolate and identify these pathogens from clinical samples received routinely in our Bacteriology Laboratory and to analyze their antibiotic susceptibility patterns. Methods: In this cross-sectional study, the first 100 NLF-GNB strains isolated consecutively from 1218 clinical samples were included through convenience sampling. The samples were processed using standard microbiological techniques. Results: The most common isolate was Pseudomonas aeruginosa followed by Acinetobacter spp, Proteus spp, Shigella spp, Salmonella typhi, Providencia spp., and Morganella spp. P. aeruginosa and Acinetobacter spp. isolates were found to exhibit high susceptibility toward Colistin and Imipenem. Proteus spp . exhibited high sensitivity toward Imipenem, Aminoglycosides, Ceftazidime, and Cefepime. All Providencia isolates were susceptible to Amikacin, Cefepime, and Ceftriaxone. The only isolate of Morganella spp. was found to be susceptible to Amikacin, Cefepime, Ceftazidime, Piperacillin tazobactam, Ciprofloxacin, Imipenem, and Aztreonam. Shigella isolates exhibited very high susceptibility toward Imipenem followed by Gentamicin and Ceftazidime. All the isolates of S. typhi exhibited susceptibility toward Imipenem, Piperacillin tazobactam, Ceftazidime, Ceftriaxone, Cefoperazone sulbactam, and Chloramphenicol. 24% of test isolates were found to be Multidrug resistant. Conclusion: Antimicrobial surveillance is needed to implement appropriate timely interventions to restrict the spread of multidrug-resistant clones. Strict infection prevention and control practices, with judicious antibiotic prescription policy, may help in tackling this problem by obviating the selection pressure.


INTRODUCTION
Aerobic non-fermenting Gram-negative bacilli (NFGNB) are a taxonomically diverse group of bacteria that are either not capable of utilizing carbohydrates as an energy source or degrading them via oxidative pathway [1][2][3].
NFGNB (Pseudomonas, Acinetobacter, Burkholderia, Stenotrophomonas, etc.) constitute about 15% of all clinical bacterial isolates. NFGNBs are emerging as important health-care-associated pathogens in the current scenario. Hospital strains are found to exhibit multidrug resistance (MDR). They have been incriminated in infections, such as septicemia, meningitis, pneumonia, urinary tract infections, and surgical site infections. NFGNBs are innately resistant to many antibiotics and are known to produce extended-spectrum ß-lactamases (ESBL) and Metallo ß-lactamases [4][5][6].
Apart from NFGNB, there is another group of non-lactose fermenters capable of utilizing glucose (Proteus, Salmonella, Shigella, Morganella, Providencia, etc.). These NLF-GNB are Notorious pathogens reportedly acquiring multiple drug resistance alarmingly and emerging as a public health threat.
MDR has been emerging rapidly and consistently in these groups of pathogens driven by selection pressure due to inappropriate irrational drug therapy. This has resulted in treatment failures leading to an extended hospital stay, health complications, and a significant rise in morbidity and mortality. The study of antibiotic susceptibility patterns of isolates is imperative in formulating a strategy for prompt and appropriate therapy and also plays a crucial role in the prevention and control of the disease. Continual consistent surveillance and monitoring of local antimicrobial resistance trends is a prerequisite for implementing rational measures and updating the therapeutic guidelines [4,[6][7][8].
There are very few studies from India wherein the various NLF-GNBs, isolated from patients' samples, have been identified and their clinical significance assessed. Hence, this cross-sectional study was undertaken to identify the various non-lactose fermenters isolated from the patients attending our hospital to assess their clinical significance and antimicrobial susceptibility pattern.

METHODS
This cross-sectional study was conducted in the Department of Microbiology, Government Medical College, Datia (MP), India, after obtaining clearance from Institutional Ethics Committee.

Mehta and Diwakar
samples processed as per the standard protocol in the duration between October 1, 2018, to April 30, 2020, whichever is earlier were included in the study.
The NLF-GNB isolates found to be contaminants or commensals concerning for the respective samples were excluded from the study. For e.g., Pseudomonas aeruginosa or Proteus spp. if isolated from stool culture were not included in the study, being a part of commensal flora of gastrointestinal tract.
Isolation and identification of bacterial isolates were done using standard microbiological techniques. The antimicrobial susceptibility test of all the test isolates was performed by the Kirby-Bauer Standard disc diffusion method and results were interpreted according to the CLSI 2018 guidelines [9][10][11].
As evident in Fig. 1 Malini et al. 2009, andGrewal et al. 2020. But in the majority of studies conducted in the recent past, the test isolates were derived predominantly from pus and respiratory samples [1,7,12,15,16].
But unlike our study, in few similar studies conducted by Sharma and Pant, 2017, Shah and Vaghela, 2018, and Paul and Borah, 2020, Acinetobacter spp. was the most common isolate followed by P. aeruginosa [4,22,23].
Out of the 100 samples yielding the test isolates, polymicrobial growth was seen in 22 samples where the NLF-GNB were isolated along with other organisms. None of the samples yielded more than one NLF isolate.
The overall isolation rate of NLF isolates from the total 1218 clinical samples processed came out to be 8.2%. P. aeruginosa (4%) exhibited the highest isolation rate followed by Acinetobacter (2%). The samplewise and organism-wise isolation rates are depicted in Tables 1 and 2.
As evident in Table 3, P. aeruginosa was more common in pus, ear swabs, and urine contributing to 80% of its isolates, while less common in blood, body fluids, sputum, and throat swabs. This finding is in concordance with other similar studies conducted by Reddy [27].
Out of three isolates of Salmonella typhi, two were obtained from stool culture and one from blood culture.
Shigella spp. being an enteropathogen, fecal specimens are the most preferred samples for culture. In our study too, all the six Shigella spp. isolates were obtained from stool culture as in the majority of similar studies [28][29][30][31][32][33].
Out of two isolates of Providencia spp., one each was isolated from urine and blood cultures. In the study period, we reported only one isolate of Morganella spp. from a urine sample.
In a study by Leulmi et   Proteus mirabilis Proteus vulgaris Acinetobacter spp.
Providencia spp. A. baumannii is gaining more importance as a nosocomial notorious pathogen due to its potential to form a biofilm, which accounts for its outstanding antibiotic resistance and high virulence.
Proteus mirabilis strains are generally found to be more susceptible to antimicrobials than Proteus vulgaris and other Proteus species. P. mirabilis has got intrinsic resistance to nitrofurantoin and tetracycline which could be used as an identification marker. However, it is generally susceptible to the Carbapenems, Penicillins, Aminoglycosides, and Cotrimoxazole [42].  [46]. P. penneri is generally found to be more resistant to penicillins than P. vulgaris, and its susceptibility pattern resembles more with Morganella morganii than with other Proteus spp. M. morganii and P. stuartii isolates also exhibited high resistance to ticarcillin, gentamicin, and chloramphenicol. More than 61% of isolates were Multidrug resistant strains (resistant to at least 3 groups of antibiotics). 15% of the isolates were ESBL producers with no significant difference among various species.
In the present study, Shigella spp. isolates exhibited very high susceptibility toward Imipenem followed by Gentamicin, Ceftazidime, Cefoperazone sulbactam, Piperacillin tazobactam, and Ciprofloxacin and very less susceptibility toward Cefepime and Co-trimoxazole.
While comparing the resistance patterns between two studies by Mamtha et al. in 2007 and 2012 in the same region, all Shigella isolates were found to be resistant to nalidixic acid and a marked increase in resistance was observed. Resistance to ciprofloxacin increased from 30% to 87%, norfloxacin from 20% to 83%, ampicillin from 63% to 100%, tetracycline from 74% to 84%, and Co-trimoxazole from 79% to 90%. However, there was a significant decrease in resistance against gentamicin and amikacin from 71% to 40% and 45% to 5%, respectively [32,47].
In both the studies, Shigella isolates exhibited high sensitivity toward 3GC which is in accordance with our study. Similar findings were reported by Srinivasa et al. 2009 [31].
Shigellae may be susceptible to the aminoglycosides in vitro, but not in vivo due to poor penetration of the intestinal mucosa when given orally [48].
In addition to some fluoroquinolones, pivmecillinam (amdinocillin pivoxil) and ceftriaxone are the only antimicrobials found to be effective in the treatment of MDR strains of Shigella in all age groups. However, Azithromycin can be considered as an alternative drug among adults. However, these antibiotics should be used only when local strains are resistant to Ciprofloxacin [48].  [49,53].
It is observed that many of the isolates resistant to Nalidixic acid (NARST) were found to exhibit in vitro susceptibility to fluoroquinolones. But as per CLSI guidelines, such strains (NARST) should be considered Fluoroquinolone resistant as Nalidixic acid is a surrogate marker to predict fluoroquinolone treatment failure.
The resistance against fluoroquinolones like Ciprofloxacin is following an increasing trend due to the selective pressure by its unrestricted injudicious usage in typhoid therapy. Nalidixic acid resistance among Salmonella spp. is rapidly increasing in India. However, the consistent use of Ciprofloxacin as the mainstay treatment of typhoid in NA resistant cases has led to a steady rise in MIC along with further mutations at the same locus which has resulted in the emergence of completely resistant strains.
Several studies in the recent past have shown a re-emergence of susceptibility of S. typhi toward the first-line antibiotics. This could be due to their inconsistent usage by clinicians over the last decade who are preferring newer antimicrobials over them resulting in the withdrawal of selection pressure.
In the present study, 24% of NLF-GNB test isolates were found to be Multidrug-resistant. In a hospital setting, Multidrug-resistant or panresistant strains may transmit from one patient to another through the hands of health-care workers or via environmental contamination. These notorious pathogens are the potential reservoirs of resistance genes that could be transferred to other bacterial strains. The high levels of β-lactamase production and multidrug resistance of the isolates is an emerging public health threat globally. Environmental surveillance, searching for asymptomatically colonized persons through screening of patients as well as health-care workers, and using molecular epidemiology should be the requisite strategy for investigating the clusters of infection with pan-resistant organisms. These wild strains have great potential to survive in the hospital environment so improved antibiotic stewardship and infection control measures will be needed to inhibit the emergence and spread of multidrug-resistant NFGNB in the health-care setting [27,43,54].
In the current scenario due to rapidly emerging multiple drug resistance, the polymixins (polymyxin B and colistin) are being used frequently as the last line therapeutic option. However, the clinicians should restrict the use of the reserve drugs in exceptional conditions only [19].

CONCLUSION
The present study highlighted the fact that non-fermenter Gramnegative bacilli like P. aeruginosa and Acinetobacter spp. along with the other non-lactose fermenter, GNBs like the Proteae family, S. typhi, and Shigella spp. had emerged as important pathogens causing serious infections in the community as well as hospital settings. Infections caused by these notorious pathogens are difficult to treat as they are rapidly acquiring resistance to the commonly used antibiotics. Ever-increasing problem of antibiotic resistance has been worsened by the slow pace of research regarding the development of newer antimicrobial molecules.
Antimicrobial surveillance is needed to implement appropriate timely interventions to restrict the spread of multidrug-resistant clones. Strict infection prevention and control practices, with judicious antibiotics prescription policy, may help in tackling the emerging threat of multiple drug-resistant bugs by obviating the selection pressure. Appropriate judicious selection and rotation/cycling of antibiotics guided by the knowledge of their susceptibility profiles is of utmost importance.
The implementation of an antibiotic policy at the hospital level for the control and restriction of injudicious antimicrobial use is imperative in managing nosocomial infections.
This is the need of the hour to develop therapeutic protocols guided by susceptibility profiles for tuning antibiotic therapy regimens to minimize the dissemination of antibiotic-resistant pathogens. Furthermore, the isolation of infected or colonized patients is of paramount importance.
Global antimicrobial susceptibility surveillance systems should also focus on the prevalence of multidrug-resistant/pan-resistant organisms, rather than restricting to just resistance rates to individual antibiotics so that the global impact of this problem could be assessed.

AUTHOR'S CONTRIBUTIONS
All the authors have made substantial contributions to conception, design, acquisition of data, analysis, interpretation, drafting manuscript, and have given final approval of the version to be published.

CONFLICTS OF INTEREST
None declared (by all authors).