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\title{Comparison of CHROMagar Orientation versus CLED (cystinelactose-electrolyte-deficient) Agar, VITEK-XL and MALDI-TOF in a Tertiary Laboratory Setting Processing Urine Culture Samples at Dr. Lal Path Labs, Delhi}
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             \author[1]{Puneeta  Singh}

             \author[2]{Shalabh  Malik}

             \author[3]{Vandana  Lal}

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\date{\small \em Received: 15 June 2021 Accepted: 5 July 2021 Published: 15 July 2021}

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\begin{abstract}
        


To comparatively assess the performance and evaluate the advantages of CHROMagar orientation vs. CLED agar for the detection and enumeration of the most common yeast, gram-positive and gram-negative urinary tract pathogens.Methods: Five hundred and eighty-seven fully characterized isolates (372Gram-negative bacteria, 106 Gram-positive bacteria, 13 Candida spp. and 96 mixed culture) were used to test for accuracy of organism identification. To assess isolation rates of common urine isolates and ability to detect mixed cultures, 2500urine samples were tested by parallel inoculation on the two best-performing media, CHROMagar orientation and CLED.Results: Of the 2550 urine specimens, 587(23.1%) yielded positive cultures, of which 491(83.6%) were pure cultures and 96(16.4%) were mixed cultures. CLED, CHROMagar orientation agar gave detection rates of 78.8% and 99.4% respectively.

\end{abstract}


\keywords{urine culture, CHROMagar orientation, CLED agar, presumptive identification.}

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\let\tabcellsep& 	 	 		 
\section[{Introduction}]{Introduction}\par
rinary tract infections (UTIs) are the second most common infections, only after respiratory tract infections. Conventionally, Blood agar (BA), Mac Conkey agar (MAC), and Cysteine Lactose Electrolyte Deficient (CLED) medium used routinely for processing of urine samples \hyperref[b0]{[1]}. Several chromogenic media are now available, which are used to allow more specific and direct differentiation of bacterial colonies on the primary plate itself \hyperref[b0]{[1]}\hyperref[b1]{[2]}\hyperref[b2]{[3]}\hyperref[b4]{[4]}\hyperref[b5]{[5]}\hyperref[b6]{[6]}\hyperref[b7]{[7]}\hyperref[b8]{[8]}\hyperref[b9]{[9]}. The following study conducted to evaluate the advantages of CHROMagar orientation over isolation of most common urine isolates (E.coli, Enterococcus faecalis, E. faecium, Staphylococcus aureus, streptococcus sps Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Proteus mirabilis and Enterobacter species\& Candida species) represent a global threat to human health \hyperref[b1]{[2]}\hyperref[b2]{[3]}\hyperref[b4]{[4]}\hyperref[b5]{[5]}\hyperref[b6]{[6]}. Urine samples contribute greatly to the daily workload of a microbiology laboratory, CHROMagar orientation has the advantage of being technically simple, rapid and cost-effective method for the diagnosis of urinary tract infections as compared to the conventional methods \hyperref[b6]{[6,}\hyperref[b9]{9]}.\par
In our lab continually, we strive to streamline and improve their urine culture algorithms because we received high volumes of urine specimens and the modest numbers of different species of bacteria that are ultimately considered clinically significant. In the current study, we quantitatively measured the impact of CHROMagar orientation media used as tools in the early differentiation and identifying of bacterial isolates from urine specimens. We have evaluated the CHROMagar orientation, a newly introduced chromogenic medium, for its utility as primary isolation and identification medium for correctly identify more-frequently occurring bacteria and yeasts organism groups on primary culture with no further testing or a minimum number of confirmatory tests. Substrates present in chromogenic media target specific classes of enzymes produced by certain bacteria and yeasts \hyperref[b6]{[6]}.\par
CHROMagar orientation media may facilitate improved sensitivity of identifying of some Gram-positive cocci (e.g., Enterococci) in mixed cultures with Enterobacteriaceae. They may promote the uniform interpretation of urine culture plates by less experienced bench technologists \hyperref[b4]{[4]}. The purpose of the current study for implementing CHROMagar orientation could be realized by use as the primary medium for urine culture and reduce workload of test, turnaround time, and labor costs. 
\section[{II.}]{II.} 
\section[{Material and Methods}]{Material and Methods}\par
An evaluation of two commercial media undertaken using isolates of known identity to assess the level of accuracy of presumptive identification. Subsequently, an assessment of the two bestperforming media in our laboratory adopting a standardized protocol to determine isolation rates and detect mixed cultures. The study was conducted at Dr. Lal Path Labs largest clinical microbiology laboratory in North India, which collectively processes approximately 500,000 urine specimens per year. 
\section[{a) Media preparation, inoculation, and incubation}]{a) Media preparation, inoculation, and incubation}\par
CHROMagar orientation (CO) (CHROMagar company, Paris, France) and CLED agar (Hi-Media Laboratories Pvt. Ltd. Mumbai-400086, India)were obtained as a dehydrated powder form. All culture petri plates were prepared in house by following manufacturer's instructions and recommendations. Every fresh batch of media was tested for its ability to support the growth of Escherechia coli ATCC (25922) to ensure the quality of the media. Urine samples were inoculated onto CLED agar and CO medium plates using a calibrated 0.001-ml loop and streaked manually. The inoculated plates (CLED agar or CO medium) were incubated at 37°Cover night (18-24 hrs) and examined at the intervals of 6hrs 12hrs, 18hrs 24hrs, and 48 hrs. Samples showing significant bacterial growth were further recorded. This study was carried out in the Department of Microbiology, at Dr. Lal path Labs, Delhi from 1 st November 2020 to 31 st January 2021. In total, 2,550 routine urine samples (predominantly in boric acid) received in our laboratories during a three months in 2020-2021, from both hospital and general practice, were included in the study. 
\section[{b) Plate reading}]{b) Plate reading}\par
CHROMagar media utilize synthetic chromogenic enzyme substrates to specifically target pathogenic species (or groups of species) based on their enzyme activity. Such enzyme activity is never completely species-specific, necessitating complementary enzyme substrates and selective agents.\par
For the purpose of our study, plates were recorded according to colonial morphology. The numbers of each colony type were also recorded to support the evaluating of the contributing organism counts of mixtures. The organism obtained from the CHROMagar orientation agar media was of different colors. E.coli gives dark pink to reddish color colony, Klebsiella, Enterobacter, Citrobacter ? metallic blueProteus ? brown halo, Pseudomonas ? greenish translucent, Acinetobacter baumanii ?cream, round translucent, bacterial isolates S. aureus ? golden, opaque, small, S. saprophyticus ? pink, opaque, small However, MALDI-TOF techniques were used to confirm the identification of organism at species level of yeast and bacterial isolates. 
\section[{c) Presumptive identification}]{c) Presumptive identification}\par
Presumptive identification of bacterial growth was done on CHROMagar orientation agar according to colony morphology and colour as depicted by the manufacturer (Figure1, 2) whereas when using CLED agar plates other tests and procedures were often required to differentiate between organisms. The final identification of the isolates was done using standard identification protocol such as VITEK -2XL (Biomerieux, France) and MALDI-TOF (Bruker Daltonics) as appropriate for the isolates. 
\section[{d) Statistical methods}]{d) Statistical methods}\par
For the study, data were collected and entered into an Excel spreadsheet. 
\section[{III.}]{III.} 
\section[{Results}]{Results}\par
The present study undertaken to validate the usefulness of CHROMagar orientation UTI agar as a primary urine culture medium for its rate of isolation and presumptive identification of uropathogens in comparison to CLED in a Dr. Lal Path Labs. Out of the 2550 urine samples processed, 587samples were positive (23.1\%) and 1963 samples (76.9\%) were negative.\par
Among the 587 positive samples Escherichia coli was the predominant Gram-negative isolate and Enterococcus faecalis was the predominant Grampositive isolate. This study included (587) positive isolates consecutively collected from both male and female population aged 0-100 midstream and/or catheter catch urine samples obtained from patients having bacteriuria in urinary tract infection. Based on data extracted from our Laboratory Information System from 2019-2020, the use of CHROMagar orientation medium resulted in a 28\% reduction in workload for additional procedures such as Gram stains, subcultures, identification panels, agglutination tests, and biochemical tests and MALDI-TOF.\par
In the present study, CHROMagar Orientation was evaluated as a direct isolation medium for clinical specimens. 587 positive urine samples were tested by parallel inoculation on CHROMagar Orientation and on other reference media, CLED agar.\par
The analysis of the data obtained from CLED, CHROMagar Orientation agar for the detection of different bacteria, result indicated that the growth pattern of the uropathogens were different. It could be due to the different constituents and properties of the media. From the study, it observed that the growth of organism over the media was according to the characteristics of the media. Mixed cultures were differentiate easily on CHROMagar orientation. On CLED agar lactose fermenting organism grows which gives yellow color colonies. However, The overall impression of the color changes produced on CHROMagar orientation media by E. coli (pink-red) which was the predominant species (32.5\%). All these isolates grew on CHROMagar Orientation in reddish colonies and were very easy to distinguish. Since E. coli is responsible for many of the UTI in nosocomial patients Klebsiella spp., (blue) and the Acinetobacter spp. should be added to the list of gram-negative microorganisms that can be presumptively differentiated directly on CHROMagar Orientation. They grew in nontransparent, white, entire-edge colonies. These strains were very distinct from Pseudomonas isolates, which grew in diffuse, yellow-togreen colonies with serrated edges that they were distinct and easy to perceive. Similarly, tryptophan is also present in the medium to detect members of the Proteus group, which generates a diffuse brown coloration background because of tryptophan deaminase production.\par
In the study gram positive bacteria were also isolated as one chromogenic substrate cleaved by ßglucosidase possessed by Enterococci resulting in formation of turquoise colonies and S.aureus gives golden yellow color colonies.\par
The results of the study to CHROMagar Orientation differentiate the most commonly encountered gram-negative pathogens gram-positive and fungal uropathogens because of color and morphology alone compared to CLED agar. CHROM agar supported the growth of all common routine urinary isolates can be recommended as a primary plating medium for recovery of uropathogens and the ease of distinguishing when multiple probable pathogens were present (Figure1).  \hyperref[tab_1]{2} respectively.\par
For presumptive identification of bacterial species by colony characteristics on primary culture of 491 bacterial and yeast isolates, 491(100\%), 488(99.4\%), 484 (98.5\%) and 388(79\%) could be differentially identified on MALDI-TOF, Vitek2-XL,CHROMagar Orientation and CLED agar respectively. The rate of presumptive identification of the isolates was found significantly higher on CHROMagar Orientation agar than CLED agar as primary urine culture medium (Table \hyperref[tab_0]{1}; Figure \hyperref[fig_1]{2}). E. coli was the leading bacteria isolated from 171 (34.8\%) samples followed by Klebseilla pneumoniae 89 (18.1\%), Enterococcus spp. 73 (14.8\%), Pseudomonas aeruginosa 54 (10.9\%), Acinetobacter spp. 21 (4.3\%), Staph.aureus 16 (3.3\%), Proteus mirabilis 13 (2.6\%), Candida spp. 13 (2.6\%), Enterobacter spp. 9 (1.8\%), Staph. saprophyticus 11 (2.2\%), and Streptococcus agalactiae 6 (1.2\%) respectively.\par
Presumptive identification of mostly gramnegative and gram-positive common uropathogens such as E.coli, K.pneumoniae, Proteus, Pseudomonas, Morganella morganii, and Enterococci spp. was correct on the CHROMagar media. E. coli was correctly identify in 99 to 100\% of the cases. 4-5 of total 54 isolates of Pseudomonas aeruginosa were not correctly presumptively identify on the CLED media. Six of Citrobacter spp., 9 of Enterobacter spp. isolates presumptively misidentified as E. coli on the CLED agars. The colony appearance of Serratia on the chromogenic media was either Red in 4 of the 9 isolates and 5 strains from the typical colony appearance of the Klebsiella-Enterobacter-Serratia group (i.e., blue, mucoid) as described by the manufacturers. The overall impression of the color changes produced on chromogenic media by E. coli, Enterococci, Klebsiella spp., Serratia spp., and the Proteus-Morganella-Providencia group that are distinct and easy to perceive. All the isolates of Enterococcus faecalis and E. faecium correctly identified at genus level and were easily distinguished from Streptococcus agalactiae isolates. Staphylococcus saprophyticus isolates were easy to identify only on the CHROMagar orientation medium whereas in CLED agar S. saprophyticus and E. faecalis have shown same colony characteristics (Figure \hyperref[fig_1]{2}).All of the gram-positive isolates were misidentified on CLED agar.\par
In this study, a total 21isolates of Acinetobacter spp. we presumptively identified 18 isolates of Acinetobacter baumanii on CHROM agar whereas species level differentiation of Acinetobacter spp were showed difficulty in CHROMagar. Similarly remaining 3 isolates of Acinetobacter spp. were identify as A. junii (2), and A. iwofii (1) by MALDITOF however, in CLED agar Acinetobacter spp were poorly identified. The identification results obtained from the Vitek-2XL system were not consistent with those from the MALDI-TOF for few Candida spp. Furthermore, identification results of 10 Candida spp. isolates from the MALDI-TOF system were the same as those from the Vitek 2 system (data not listed). In this study, we evaluate the identification performance of MALDI-TOF MS for identification of enteropathogens and yeast isolates with a lower identification error rate, MALDI-TOF MS has better performance than VITEK 2 in identifying yeast found routinely in the clinical laboratory.  Table \hyperref[tab_1]{2} Shows the rate of presumptive identification of polymicrobial growth in different culture media. All 139 (100\%) polymicrobial growths distinctly identified only on Chromagar Orientation agar medium, in contrast except in a single case consisting of E. coli and Proteus mirabilis, no mixed bacterial growths could be identified on CLED agar media. The detection of Gram positives and yeasts organisms diminishes in the presence of increasing numbers of Gram-negative organisms, because of the white or colorless appearance of the colonies on the CHROMagar orientation media for Gram-positive organisms and yeasts, CHROMagar performed better than other UTI medium such as CLED (Table  {\ref .}2).\par
In our study, CHROMagar showed a superior differentiation of mixed cultures because different   In this study, we evaluated CHROMagar Orientation from The CHROMagar Company [Paris, France] for routine diagnosis of bacteriuria at our laboratory concerning isolation frequency and presumptive identification of urine isolates. CLED (cysteine-, lactose-, and electrolyte-deficient) agar, were used as the reference media. We also compared the interval of 6hrs incubation to 48hrs of incubation; to our knowledge, this has not done previously. The media evaluation were listed in (Table \hyperref[tab_2]{3}). According to the technical data, when the total number of isolates recovered from both of the media was compared to the number of isolates growing on the individual media types after an interval of6-48 hours incubation period. The percentage for CHROMagar Orientation media shows approximately 20\% high in colony count in 13-18 hours incubation that was evident in the present study. Although incubation longer than overnight (up to 24-48 hours) does not significantly increase the yield of common, urine isolates on CHROMagar orientation or traditional media CLED. In this study, we found that most common gram-negative isolates such as E.coli, K.pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa and Acinetobacter spp. in 0-6 hrs incubation period no growth were seen in both media (Table \hyperref[tab_2]{3}, Figure \hyperref[fig_4]{4}). However, 7-18 hrs incubation period showed that CHROMagar Orientation performing better growth than CLED whereas after 18hrs incubation, there growth pattern were similar in both media. CHROMagar Orientation, performed better growth of Gram-positive isolates in a short incubation period and easily identified after 18 hrs incubation (Table \hyperref[tab_2]{3}). Similarly, CHROMagar Orientation given the best result for isolation of yeast species in 18-24hrs incubation period (Table-3).  
\section[{Discussion}]{Discussion}\par
Every clinical microbiology laboratory's daily workload of urine cultures account for a diagnosis of urinary tract infection because only 20 to 30\% of urine samples result in significant growth \hyperref[b0]{[1,}\hyperref[b2]{3]}. Therefore, any new medium or method with the ability to streamline urine culture processing in a meaningful way, such as reducing technologist workload, improving result turnaround times (TATs), or reducing laboratory costs, would be welcomed and has the potential to have considerable laboratory impact. Our study confirmed the superiority of CHROMagar orientation over CLED agar in detecting mixed cultures, Gram-positive organisms, and yeasts; these results corroborate earlier studies \hyperref[b1]{[2]}\hyperref[b2]{[3]}\hyperref[b4]{[4]}\hyperref[b5]{[5]}\hyperref[b6]{[6]}.\par
Traditionally conventional media like Blood agar (BA) the majority of urine isolates as an enriched medium but its performance in the identification of bacteria is very deficient. Similarly, differentiation of lactose fermenter and non-fermenter is possible on MAC and CLED agar. Moreover, none of these media singly or in combination can support the growth and identification of possible urine isolates \hyperref[b0]{[1,}\hyperref[b7]{7]}. As a result, further species identification necessitates subculture or divergent tests with longer reporting time and cost. The present findings were in concordance with the findings of \hyperref[b1]{(Aspevall et al., 2002)} observed that the CHROMagar Orientation media tested in this study was better than CLED agar. A similar observation was also reported by \hyperref[b4]{(Fallon et al., 2003)} using BBL CHROMagar, UTI medium, or CPS ID2 chromogenic agar, as a replacement for Cystine Lactose Electrolyte Deficient agar (CLED) would improve the detection rate of contaminated urine samples. "A cost comparison of the agars suggests that as the use of chromogenic agar in laboratories increases, the purchase cost is decreasing" \hyperref[b4]{(Fallon et al., 2003)} \hyperref[b6]{[6]}.\par
In the present study, the time interval between plating and final organism identification was decreased on CHROMagar orientation and it was seen that were evident within 18-hours versus CLED using the entire required standard microbiological tests; it was an average of 38 hours. Using CHROMagar orientation, clinically significant cultures required less hands-on time. Similarly in a study by Bajoria et al., concluded that conventional media requires 24-48 hours to give positive results \hyperref[b2]{[3]}. Articles reported the effect of incubation time on results of urine culture on traditional media \hyperref[b1]{[2]}. All agree that common urine isolates detected after overnight incubation and that a longer incubation time is required for the detection of yeasts.\par
Hence, it concluded that the cost comparison of the agars suggests that the use of CHROMagar orientation in laboratories increases, the purchase cost is decreasing due to the needs for repeat samples, and avoided antimicrobial therapy because of improved mixture detection \hyperref[b0]{[1,}\hyperref[b1]{2]}. In a few studies comparing CHROMagar Orientation media with traditional ones, its advantages including a 20\% reduction in time for identification, reduction in workload \hyperref[b5]{[5,}\hyperref[b6]{6,}\hyperref[b8]{8]}. When using traditional media requires a great deal of experience for presumptive identification of isolates, whereas CHROMagar media, is easier, requiring less training and interpreted by personnel with less experience in microbiology. Thus, the use of CHROMagar Orientation media may improve the quality of urine culture by contributing to a uniform interpretation of urine culture plates by the different personnel engaged in this task at the laboratory. All these factors have a direct impact on ultimate cost reduction. Our data support the findings of these investigators \hyperref[b1]{[2]}\hyperref[b2]{[3]}\hyperref[b4]{[4]}\hyperref[b5]{[5]}\hyperref[b6]{[6]}\hyperref[b7]{[7]}\hyperref[b8]{[8]}. Also, MALDI-TOF MS showed to be simple, rapid, and accurate tool for the identification of enteropathogens and rare yeast species, At the same time the Vitek 2 XL system is a popular commercial method commonly used in clinical microbiology laboratories for bacterial identification.\par
Most the isolates analyzed in our study largely commonly found pathogens, and the construction of the MALDI-TOF MS database may offer higher identification accuracies for these pathogens. Additionally, MALDI-TOF MS dramatically shortened identification time from 6-8 hours to just a few minutes. However, MALDI-TOF MS made no errors at the genus and species level while VITEK -2XL made 0.6\% errors at the species level of rare yeast species \hyperref[b10]{[10,}\hyperref[b11]{11]}.\par
V. 
\section[{Conclusion}]{Conclusion}\par
CHROMagar Orientation provided the highest overall organism recovery rates, convenient for rapid identification, and the greatest ability to detect mixed cultures. The use of CHROMagar orientation medium as a replacement for Cystine Lactose Electrolyte Deficient (CLED) agar would improve the detection rate of contaminated urine samples and has the potential to streamline urine culture processing in a meaningful way, such as reducing technologist workload, improving result of turnaround times and reducing costs. It would improve identification that helps to distinguish species, facilitating the monitoring of bacterial resistance in support of the national antibiotic strategy.\par
Ethical Approval: It is not applicable. 
\section[{Conflicts of interest:}]{Conflicts of interest:}\par
There are no conflicts of interest.\begin{figure}[htbp]
\noindent\textbf{1}\includegraphics[]{image-2.png}
\caption{\label{fig_0}Figure 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{2}\includegraphics[]{image-3.png}
\caption{\label{fig_1}Figure 2 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-4.png}
\caption{\label{fig_2}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3}\includegraphics[]{image-5.png}
\caption{\label{fig_3}Figure 3 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{4}\includegraphics[]{image-6.png}
\caption{\label{fig_4}Figure 4 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-7.png}
\caption{\label{figure7}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{}\includegraphics[]{image-8.png}
\caption{\label{figure8}}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{1} \par 
\begin{longtable}{P{0.2894866732477789\textwidth}P{0.00839091806515301\textwidth}P{0.13845014807502468\textwidth}P{0.14600197433366238\textwidth}P{0.14768015794669298\textwidth}P{0.11999012833168805\textwidth}}
\multicolumn{2}{l}{Uropathogens N=491}\tabcellsep CHROMagar orientation N=484 (98.6\%)\tabcellsep MALDI-TOF Identification N=491(100\%)\tabcellsep VITEK-XL identification N=488 (99.4\%)\tabcellsep CLEDagar N=388 (79\%)\\
\multicolumn{2}{l}{Escherichia coli (171)}\tabcellsep 171 (100\%)\tabcellsep 171 (100\%)\tabcellsep 171 (100\%)\tabcellsep 169 (98.8\%)\\
\multicolumn{2}{l}{Klebsiella pneumonia (89)}\tabcellsep 89 (100\%)\tabcellsep 89 (100\%)\tabcellsep 89 (100\%)\tabcellsep 87 (97.8\%)\\
\multicolumn{2}{l}{Proteus mirabilis (13)}\tabcellsep 13\tabcellsep 13 (100\%)\tabcellsep 13 (100\%)\tabcellsep 13\\
\multicolumn{2}{l}{Enterobacter spp. (9)}\tabcellsep 9 (100\%)\tabcellsep 9 (100\%)\tabcellsep 9 (100\%)\tabcellsep 0 (0\%)\\
\multicolumn{2}{l}{Citrobacter koseri (6)}\tabcellsep 6 (100\%)\tabcellsep 6 (100\%)\tabcellsep 6 (100\%)\tabcellsep 0 (0\%)\\
Pseudomonas\tabcellsep aeruginosa\tabcellsep 54 (100\%)\tabcellsep 54 (100\%)\tabcellsep 54 (100\%)\tabcellsep 49 (90.7\%)\\
(54)\tabcellsep \tabcellsep \tabcellsep \tabcellsep \tabcellsep \\
\multicolumn{2}{l}{Acinetobacter spp. (21)}\tabcellsep 18 (85.7\%)\tabcellsep 21 (100\%)\tabcellsep 21 (100\%)\tabcellsep 9 (42.8\%)\\
\multicolumn{2}{l}{Serratia marcescens (9)}\tabcellsep 9 (100\%)\tabcellsep 9 (100\%)\tabcellsep 9 (100\%)\tabcellsep 4 (44.4\%)\\
\multicolumn{2}{l}{Enterococcus faecalis (52)}\tabcellsep 52 (100\%)\tabcellsep 52 (100\%)\tabcellsep 9 (100\%)\tabcellsep 43 (58.9\%)\\
\multicolumn{2}{l}{Enterococcus faecium (21)}\tabcellsep 21 (100\%)\tabcellsep 21 (100\%)\tabcellsep 21 (100\%)\tabcellsep 9 (42.8\%)\\
\multicolumn{2}{l}{Staphylococcus aureus (16)}\tabcellsep 16 (100\%)\tabcellsep 16 (100\%)\tabcellsep 16 (100\%)\tabcellsep 0 (0\%)\\
Staphylococcus saprophyticus (11)\tabcellsep \tabcellsep 11(100\%)\tabcellsep 11 (100\%)\tabcellsep 11 (100\%)\tabcellsep 0 (0\%)\\
\multicolumn{2}{l}{Streptococcus agalactiae (6)}\tabcellsep 6 (100\%)\tabcellsep 6 (100\%)\tabcellsep 11 (100\%)\tabcellsep 0 (0\%)\\
Candida spp.(13)\tabcellsep \tabcellsep 9 (69.2\%)\tabcellsep 13 (100\%)\tabcellsep 10 (76.9\%)\tabcellsep 5 (38.4\%)\end{longtable} \par
 
\caption{\label{tab_0}Table 1 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{2} \par 
\begin{longtable}{P{0.5107375271149674\textwidth}P{0.1843817787418655\textwidth}P{0.15488069414316705\textwidth}}
Uropathogens N=96 (16.4\%)\tabcellsep CHROMagar orientation N=96 (100\%)\tabcellsep CLED agar N =74 (77\%)\\
E. coli and K. pneumoniae (23)\tabcellsep 23(100\%)\tabcellsep 22(95.7\%)\\
E coli and Enterococcus spp. (29)\tabcellsep 29 (100\%)\tabcellsep 17 (58.6\%)\\
K. pneumoniae and Pseudomonas aeruginosa (12)\tabcellsep 12 (100\%)\tabcellsep 11 (91.6\%)\\
E.coli and Pseudomonas aeruginosa (19)\tabcellsep 19 (100\%)\tabcellsep 18 (94.7\%)\\
Proteus mirabilis and E.faecalis (3)\tabcellsep 3 (100\%)\tabcellsep 0 (0)\\
Proteus mirabilis and E.coli (6)\tabcellsep 6 (100\%)\tabcellsep 6 (100\%)\\
Staphylococcus aureus and E. coli (4)\tabcellsep 4 (100\%)\tabcellsep 0 (0)\end{longtable} \par
 
\caption{\label{tab_1}Table 2 :}\end{figure}
 \begin{figure}[htbp]
\noindent\textbf{3} \par 
\begin{longtable}{P{0.1598342939481268\textwidth}P{0.002449567723342939\textwidth}P{0.06307636887608069\textwidth}P{0.07777377521613832\textwidth}P{0.08512247838616714\textwidth}P{0.08512247838616714\textwidth}P{0.08206051873198847\textwidth}P{0.18004322766570605\textwidth}P{0.014697406340057638\textwidth}P{0.09981988472622479\textwidth}}
\multicolumn{2}{l}{Incubation period}\tabcellsep \tabcellsep 0-6 hrs\tabcellsep \tabcellsep 7-12 hrs\tabcellsep \multicolumn{2}{l}{13-18 hrs}\tabcellsep \multicolumn{2}{l}{19-24 hrs}\\
\multicolumn{2}{l}{Bacterial isolates}\tabcellsep CLED\tabcellsep CHROMagar orientation\tabcellsep CLED\tabcellsep CHROMagar Orientation\tabcellsep CLED\tabcellsep CHROMagar Orientation\tabcellsep CLED\tabcellsep CHROMagar Orientation\\
\multicolumn{2}{l}{E. coli (n=145)}\tabcellsep No growth\tabcellsep No growth\tabcellsep 10 2 cfu/ml\tabcellsep 10 4 cfu/ml\tabcellsep 10 5 cfu/ml\tabcellsep \multicolumn{2}{l}{>=10 5 cfu/ml >=10 6 cfu/ml}\tabcellsep >=10 6 cfu/ml\\
\multicolumn{2}{l}{K.pneumoniae (n=85)}\tabcellsep No growth\tabcellsep No growth\tabcellsep 10 2 cfu/ml\tabcellsep 10 3 cfu/ml\tabcellsep 10 5 cfu/ml\tabcellsep \multicolumn{2}{l}{>=10 5 cfu/ml >=10 6 cfu/ml}\tabcellsep >=10 6 cfu/ml\\
\multicolumn{2}{l}{P. mirabilis(n=13)}\tabcellsep No growth\tabcellsep No growth\tabcellsep 10 2 cfu/ml\tabcellsep 10 3 cfu/ml\tabcellsep 10 5 cfu/ml\tabcellsep \multicolumn{2}{l}{>=10 5 cfu/ml >=10 6 cfu/ml}\tabcellsep >=10 6 cfu/ml\\
\multicolumn{2}{l}{P.aeruginosa(n= 54)}\tabcellsep No growth\tabcellsep No growth\tabcellsep 10 2 cfu/ml\tabcellsep 10 3 cfu/ml\tabcellsep 10 5 cfu/ml\tabcellsep \multicolumn{2}{l}{>=10 5 cfu/ml >=10 5 cfu/ml}\tabcellsep >=10 5 cfu/ml\\
\multicolumn{2}{l}{Enterococcus spp. (n=73)}\tabcellsep No growth\tabcellsep No growth\tabcellsep 10 1 cfu/ml\tabcellsep 10 4 cfu/ml\tabcellsep 10 4 cfu/ml\tabcellsep \multicolumn{2}{l}{>=10 5 cfu/ml >=10 4 cfu/ml}\tabcellsep >=10 5 cfu/ml\\
\multicolumn{2}{l}{Acinetobacter baumanii (n=21)}\tabcellsep No growth\tabcellsep No growth\tabcellsep 10 2 cfu/ml\tabcellsep 10 3 cfu/ml\tabcellsep 10 4 cfu/ml\tabcellsep \multicolumn{2}{l}{>=10 4 cfu/ml >=10 5 cfu/ml}\tabcellsep >=10 5 cfu/ml\\
Enterobacter spp.(n=9)\tabcellsep \tabcellsep No growth\tabcellsep No growth\tabcellsep 10 2 cfu/ml\tabcellsep 10 3 cfu/ml\tabcellsep 10 3 cfu/ml\tabcellsep \multicolumn{2}{l}{>=10 4 cfu/ml 10 5 cfu/ml}\tabcellsep >=10 5 cfu/ml\\
\multicolumn{2}{l}{Streptococcus agalactiae(n=6)}\tabcellsep No growth\tabcellsep No growth\tabcellsep 10 1 cfu/ml\tabcellsep 10 2 cfu/ml\tabcellsep 10 3 cfu/ml\tabcellsep \multicolumn{2}{l}{>=10 4 cfu/ml 10 5 cfu/ml}\tabcellsep >=10 5 cfu/ml\\
\multicolumn{2}{l}{S.saprophyticus (n=11)}\tabcellsep No growth\tabcellsep No growth\tabcellsep 10 1 cfu/ml\tabcellsep 10 2 cfu/ml\tabcellsep 10 3 cfu/ml\tabcellsep \multicolumn{2}{l}{>=10 4 cfu/ml 10 4 cfu/ml}\tabcellsep >=10 4 cfu/m\\
\multicolumn{2}{l}{S. aureus(n=16)}\tabcellsep No growth\tabcellsep No growth\tabcellsep 10 1 cfu/ml\tabcellsep 10 2 cfu/ml\tabcellsep 10 3 cfu/ml\tabcellsep \multicolumn{2}{l}{>=10 4 cfu/ml 10 4 cfu/ml}\tabcellsep >=10 4 cfu/ml\\
Candida (n=13)\tabcellsep spp.\tabcellsep No growth\tabcellsep No growth\tabcellsep \multicolumn{2}{l}{10 1/2 cfu/ml 10 2 cfu/ml}\tabcellsep 10 1 cfu/ml\tabcellsep 10 3 cfu/ml\tabcellsep 10 2 cfu/ml\tabcellsep >=10 3 cfu/ml\end{longtable} \par
 
\caption{\label{tab_2}Table 3 :}\end{figure}
 		 		\backmatter   			 \par
Acknowledgements: We are thankful to Dr Reena Nakra, Lab director operations National Reference Laboratory, Dr. Lal Path Labs, Delhi for providing us operational support and team Microbiology for technical assistance, Dr. Navaneeth Saxena from [r-biopharm neugen pvt ltd \& Subs-Chromogenic life science India pvt.Ltd. Hyderabad, Advices and helps in conducting the study. 			  			  				\begin{bibitemlist}{1}
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