Antimicrobial resistance surveillance among gram-negative bacterial isolates from patients in hospitals in Khartoum State,

Antimicrobial resistance (AMR) among gram-negative bacilli is a Background: global health problem. Surveillance of AMR is required to advise on empirical antimicrobial therapy. This study aimed at evaluating the frequency and the AMR patterns of gram-negative isolates from patients treated in eight hospitals in Khartoum State, Sudan. A cross-sectional laboratory-based study was conducted over a 6 Methods: months period at the Microbiology Department, Soba University HospitalKhartoum State, Sudan. All gram-negative isolates from blood, urine, wound, and sputum during the period of study were included. Identification and antimicrobial susceptibility testing were carried out for all isolates. A total of 734 Gram-negative bacilli were isolated. Results: Klebsiella (249 isolates, 34%) was the most frequently encountered one, pneumoniae followed by (153 isolates, 21%),   (123 isolates, Pseudomonas aeruginosa E.coli 17%), (75 isolates, 10%), (42 Acinetobacter baumannii Burkholderia cepacia isolates, 6%), and (28 isolates, each, (4%) Proteus mirabilis Proteus vulgaris (28 isolates, 4%), (21 Enterobacter colecaes Stenotrophomonas maltophilia isolates, 2.8%), and other gram-negative bacilli (15 isolates, 2.2%) The analysis of the antimicrobial susceptibility patterns showed that 134 (22.3%) isolates were resistant to three or more classes of antibiotics, including cephalosporins, β-lactam–β-lactamase inhibitor, quinolones, aminoglycosides and carbapenems. This high level of resistance among gram-negative bacilli in Conclusion: Khartoum state hospitals is alarming. The local health authorities should be prompted to step up infection control programs and introduce the concept of antimicrobial stewardship in Khartoum State hospitals.


me countries,
where infectious diseases are the commonest cause of hospitalization and death; and the newer antibiotics cannot be afforded 7 .

AMR surveillance is key for determining the prevalence patterns of AMR, which is fundamental for the development of national and international treatment strategies.Most of the available surveillance data are derived from developed countries; the studies conducted in developing countries are unfortunately not adequate.This surveillance study was undertaken in order to find out the different types of the AMR patterns of bacterial pathogens isolated from patients in Khartoum State, Sudan.This study may help in formulating antibiotic policies tailored to our hospitals.These data can be used as "information for action" antibiotic stewardship and interventions to optimize antibiotic prescribing practice, therefore prolongs the usefulness of existing antibiotics.


Methods


Study design and clinical strains

This is a cross-sectional laboratory based study carried out in the department of medical microbiology Soba University Hospital (SUH) and Institute of Endemic Diseases, University of Khartoum, Sudan.A total of 734 Gram-negative bacteria were isolated from patients treated in eight hospitals in Khartoum state including: two university hospitals (Soba university hospital and Bashaier); three teaching hospitals (Ibrahim Malik, Bahri and Sadabulalla) , a specialized hospital (Elfouad), and two private hospitals (Imperial and Elswedy).This study included all clinical specimens received in Soba University hospital microbiology laboratory in a period from October 2016 to February 2017 from various wards in the aforementioned hospitals including: intensive care units (ICUs), neonatal ICUs, medicine units, surgery units, pediatric units and renal units.The isolates were collected from different clinical specimens including: blood (243 isolates, 33.1%), urine (230 isolates, 31.3%),wounds (183 isolates, 25%), sputum (22 isolates, 3%), catheter tips (26 isolates, 3.4%) and body fluids (including cerebrospinal, peritoneal, pleural, acetic and synovial flu d; 30 isolates 4.2%).Microorganisms were grown on Blood, Chocolate and MacConkey agar.Then, they were identified according to standard microbiological procedures (based on colony morphology, microscopy, and biochemical tests) 8 .Quality control strains were used in biochemical tests and antimicrobial susceptibility testing [E. coli (ATCC #25922) and P. aeruginosa (ATCC #27853)].

Most gram-negative Bacilli isolates were further identified and con

R.Guanidine chloride method, as de
cribed by Alsadig et al. 9 , was used for DNA extraction followed by PCR which it was carried out using thermal cycler (analytikjena® Biometra TADVANCED, Germany), by using the following primers (Macrogen, Korea), using species-specific primers for Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii and Universal 16S rRNA primers (Table 1) the reaction was carried out in a total reaction volume of 25 μl (5μl Master mix of Maxime RT premix kit (iNtRON Biotechnology, Seongnam, Korea), 0.6 μl of forward primer, 0.6 μl of reverse primer, 2μl of DNA and 16.8 μl deionized sterile water).The cycle condition as the following: initial denaturation step at 94°C for 5-min, followed by 30 cycles of denaturation at 94°C for 45 seconds, primer annealing temperature according to the primers Table 1 for 45 seconds, followed by step of elongation at 72°C for 60 seconds and the final elongation at 72°C for 5 min 10 .The purity and integrity of each PCR product was evaluated electrophoresis in a 2% agarose gel in TBE 1X, that contain 2.5 μl of (20mg/ml) ethidium bromide at 100V for 40 min.The specific amplified product were detected by comparing with 100 base-pairs standard DNA ladder (iNtRON BIOTECHNOLOGY, Seongnam, Korea) Bands were visualized under U.V transilluminater (analytikjena® Biometra BDAcompact, Germany).The PCR product of 16SrRNA were purified and Sanger sequencing was performed by Macrogen Company (Seoul, Korea).Then nucleotides sequences of the genes 16SrRNA achieved were searched for sequence similarity using nucleotide BLAST for species identification 11 .


Antimicrobial susceptibility testing

Susceptibility testing was performed using the Kirby-Bauer disc-diffusion method; each isolate was swabbed on the Muller-Hinton agar and the antibiotic discs were placed on top and incubated at 37°C for 18-24 hours 12 .all isolates were tested against the following antibiotic disc (Mast Diagnostic): amoxycillin clavulanate (AMC) (30 μg), cefuroxime (CXM) (30 μg), cephalexin (CL) (30 μg), ceftriaxone (CRO) (30 μg), ceftazidime (CAZ) (30 μg), meropenem (MEM) (10 μg), imipenem (IPM) (10 μg), amikacin (AK) (30 μg), gentamicin (Gen) (10 μg), ciprofloxacin (CIP) (5 μg), trimethoprim-sulfamethoxazole (SXT) (25 μg), temocillin (TEM) (30 μg), azteroname (AZT) (30 μg) and nitrofrantoine (NIT) (300 μg).Results were interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines 12 .

Classification of MDR gram-negative Bacilli MDR has been considered for clinically significant gramnegative Bacilli such as E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii based on the aforementioned antimicrobial resistance definition.Classes of antibiotics used for MDR-GNB analysis were aminoglycoside (AMG), cephalosporins (CEPH), carbapenems (CARB), and fluroquinolones (FQ) as follows: bacteria that were MDR for four classes of antibiotics (AMG+ CEPH+ CARB+ FQ) and bacteria that were MDR for three classes of antibiotics (either AMG+CEPH +FQ, CARB+CEPH+FQ, AMG+CEPH+CARB, or AMG+FQ+CARB) 3 .

Cephalosporin resistance was defined as resistance to ceftriaxone and ceftazidime, except for P. aeruginosa, where only ceftazidime was used.Carbapenem resistance was defined as resistance to both meropenem and imipenem.Aminoglycoside resistance was defined as resistance to both gentamicin and amikacin, Ciprofloxacin resistance was considered an indication to fluoroquinolones resistance.


Ethical consideration

Formal permission was obtained from the managers of Soba University Hospital and the Institutional Research Ethics Committee of the Institute of Endemic Diseases, University of Khartoum, approved this study under reference number IEND_REC 12/2017.Patient consent was waived by the Research Ethics Committee.


Statistical analysis

Data were analysed using Microsoft Excel and SPSS version 20.0.Cross-tabulation was used to present the different relations between data, qualitative data were performed using a χ 2 test (significance was set at p≤0.05), which was performed to find the differences between bacterial isolates with resistance to at least one class of antibiotics by specimens (blood, urine, wound and other samples) p-values were determined for primary and secondary outcomes.


Results


Bacterial identification

Isolated Gram-negative bacilli showed different strains, including E. coli (123 isolates, 17%), K. pneumoniae (249 isolates,34%), P. aeruginosa (153 isolates, 21%), A. baumannii (75 isolates, 10%), Burkholderia cepacia (42 isolates, 6%), Proteus mirabilis and Proteus vulgaris (28 isolates, 4%), Enterobacter colecaes (28 isolates, 4%), Stenotrophomonas maltophilia (21 isolates, 2.8%) and other gram-negative bacilli (15 isol tes, 2.2%).

While isolates were distributed among the different hospital units, most of the pathogenic strains were isolated from neonatal intensive care unit (ICU) (182 isolates, 24.8%) mainly K. pneumoniae (77 isolate, 42.3%) and pediatric units (175 isolates 23.8%) the most prevalent strains were K. pneumoniae (50 isolate, 29%) and P. aeruginosa (44 isolates, 25.1), while medicine (147

l hospital units.The
istribution of different gram-negative isolates is shown in Table 2.

With regard to the distribution of the isolates among different clinical specimens, Klebsiella pneumoniae and Pseudomonas aeruginosa were isolated mainly in blood specimens 39% and 25% respectively, while K. pneumoniae and E.coli were 36% and 30% of urine samples.A. baumannii was isolated from 14% of wound specimens and P. aeruginosa was isolated from 23% of wound specimens.For more deta

am-negative bacilli among
different specimen see Table 3.


Antimicrobial resistance pattern of clinical isolates

Antibiotic resistance pattern are shown in Figure 1.Out of 734 isolates tested using the disk diffusion method, the highest percentage of resistance, in 97% and 93.5% of isolates, were found against ampicillin and cephalexin, respectively, followed by amoxicillin/clavulanic acid(90%), cefotaxime (89.7%), ceftriaxone (88.4%) and ceftazidime (79.2%).

re resistant to several antibiotic groups are shown in
Discussion

Infection with MDR gram-negative Bacilli is a major problem worldwide, associated with increased patients morbidity and mortality 17 .In Sudan, the increasing number of MDR bacteria is a real clinical challenge 18,19 .This study was undertaken; to identify the different patterns of AMR in bacterial pathogens isolated from patients treated in various wards of hospitals.

In this study, K. pneumoniae and P. aeruginosa strains were more prevalent in blood specimens while K. pneumoniae and E.coli strains were more frequently isolated in urine specimens.

K. pneumoniae is a prominent pathogen causing both hospital-acquired and community-associated infections, including bacteremia, wound i fection, pneumonia, urinary tract infections and other infections 20,21 .In this study, K. pneumoniae was the most commonly isolated organism from the blood specimens mainly in neonatal sepsis in a high rate (39.8%).Most of these strains resistant to cephalosporins and other class of antibiotics including carbapenem as reported worldwide 7, 21,22 .

E. coli is regarded as the commonest pathogen of the urinary tract, causing complicated and uncomplicated urinary tract infections 23 .In this study, the most frequently observed pathogens in urine specimens were E. coli (30%) and K. pneumoniae (36%).This finding is in harmony with the results of several other studies, including: de Francesco et al., who found that E.coli    was the commonest GNB (42.4%) isolate from urine specimens of patients with urinary tract infection 24 ;

study in Tanzania reported 38% of E.coli isolated from ur
nary specimens 25 and other studies from Pakistan and India 25,26 .

In this study, non-lactose-f

menting gra
-negative bacilli such as P. aeruginosa and A. baumannii were mostly encountered in ICU patients, these organisms were isolated in 20.4% of samples, and were found in different clinical samples such as: blood, wound and sputum.This finding agrees with a study by Vincent et al. that reported P. aeruginosa and A. baumannii were frequently isolated from ICU patients by Vince t et al. 27 ; Jitendra et al., reported that A. baumannii was the second most common pathogen in an ICU of a tertiary care center 28 .Concerning the infection type, we found that P. a ruginosa were associated with 25% of blood stream infections and 23% of wound infection, while A. baumannii mainly associated with wound infection in 14% in agree with Gales et al. 2010 29 .

Resistance of gram-negative bacilli is widespread and multidrug resistance has been reported in many studies 2,4,5 , causing challenges in the treatment of nosocomial infections.The resistance pattern was commonly reported in classes such as cephalosporins, carbapenem, aminoglycosides and quinol nes [30][31][32] .In this study, we observed high rates of resistance to extended-spectrum β-lactamases (ESBL), resistance to cephalexin, cefuroxime, ceftazidime and ceftriaxone, in addition to resistance to ampicillin and amoxicillin/clavulanic acid.

The analysis of the antimicrobial susceptibility patterns of the study isolates showed high rate of MDR organisms that were re r baumannii, being resistant to all four classes of antibiotics used in 50.6% of isolates.A total of 73.7% of A. baumannii were found to be resistant to meropenem, and 66.7% to imipenem, while in the cephalosporin class, more than 91% of the isolates were resistant.

A. baumannii also have high resistance rate to aminoglycosides and quinolones (63.2% for amikacin and 79% for both gentamicin and ciprofloxacin).This increasing resistance among A. baumannii has become a public-health issue, because this bacterium frequently causes nosocomial infections 31 .

In this study the most common clinical isolates of the Enterobacteriaceae family were K. pneumonia and E. coli.In both species, there was a high prevalence of resistance against quinolones, aminoglycosides and beta-lactams.Rates of resistance to carbapenems were alarmingly high: 22% in K. pneumoniae and 9% in E. coli.These bac eria have high resistance rate to ceftazidime and cephalexin, (80.6% and 92%, respectively).These rates are much higher than those observed in 2013 by Ali in Soba University hospital, who found that ceftriaxone and ceftazidime resistance rates ranged from 56% to 79% 21,33 .Aminoglycoside (specifically amikaci ) resistance rates among K. pneumoniae and E. coli were 16.7% and 12.1%, respectively, which is higher than those observed by Lee in 2013, who found the resistance rate to amikacin was 6.2% in K. pneumoniae and 1.3% in E. coli 30 .In this study Gentamicin resistance among both K. pneumoniae an E. coli was 53%, which is a high resistance rate.E. coli was highly resistant to quinolones like ciprofloxacin (in 66.4%), whereas K. pneumoniae was resistant in 42% of isolates.This finding is much lower than that observed by Moolchandani et al. in 2017 32 .

Pseudomonas aeruginosa was resistant to carbapenem in 22% of isolates and was highly resistant to ceftazidime (in 81% of isolates) followed by gentamicin (57.5%), ciprofloxacin (22.5%) and amikacin (9.5%).resistance to many antibiotic among P. aeruginosa was reported in many studies 4,34 .

The high level of resistance in the current study can be attributed to the unrestricted use of antibiotics in Sudanese hospitals; this injudicious use has been shown to have, an important role in increasing carbapenem resistance 32 .During this study, 134 gram negative bacilli resistant to three or four classes of antibiotics were isolated over a period of six months, which is relatively higher rate than the rate reported in a previous study also conducted in SUH over 30 months, from January 2011 to June 2013 33 .This concerning finding indicates the rapid acceleration in the rate of emergence of MDR organisms in our local sittings.Moreover, 0 bacterial strains were resistant to all available antibiotics,including meropenem.

In addition, microbiology laboratories play a crucial role against the spread of antimicrobial resistance by accurately identifying causative pathogens and detecting their antimicrobial susceptibility profi e, so as to guide the proper use of antibiotics by the care providers.Unfortunately, in Sudan the lack of reliable microbiology laboratory services adds yet another layer to the multifactorial problem of MDR organisms' epidemic in our country.Most of the laboratories in our country use the disk diffusion method for antimicrobial susceptibility testing; however, there are no policies regarding phenotypic screening for antibiotic resistance among certain organisms; resulting in that the laboratory report may not be accurate and misguide the doctors.For instance, in the disk diffusion method, if the inhibitory zone around ceftazidime was ≤22 mm, ideally this should trigger testing for the presence of ESBLs in the isolated pathogen, ecause the presence of these enzymes renders all penicillins, cephalosporins and monobactams 12 useless against that certain isolate, even if they shows susceptible results in the routine test.Yet, this extra step is not taken and only the routine susceptibility results will be reported, which may inaccurately show some antibiotics as susceptible while in reality the organism is resistant to them.Lastly, there is a limited choice of available antibiotics in Sudan; the most widely available are cephalosporins, which makes them the backbone of treating infectious diseases regardless of the isolate or its susceptibility profile.Carbapenems are believed to treat all evils, once they were reserved for the treatment of MDR organisms causing systemic infections 4 , but the recent trend led to over use them for treating infections that can be managed by less potent antibiotics, contributing to this vicious cycle that aggravate the AMR issue even more.


Conclusion

In conclusion, there was a high prevalence of gram negative bacterial pathogen associated hospital and community acquired infections, with increasing rates of resistance to available antibiotics.Strict infection control measures should be implemented, and antimicrobial stewardship should be initiated and policed to decrease the spread of MDR pathogens in Sudanese hospitals.


Open Peer Review

Current Peer Review Status:


Roberto G Melano

Public Health Ontario Laboratory, University of Toronto, Toronto, Ontario, Canada

In Methods, I suggest this modification for the very long (and unclear) sentence: "Gua idine chloride method, as described by Alsadig et al. 9 , was used for DNA extraction followed by PCR.Species-specific primers (Macrogen, Korea) were used for identification of Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii; universal 16S rRNA primers were also used for identificati

of other s
ecies (Table 1).Each amplification was carried out in a total reaction volume of 25 μl: 5μl Master mix of Maxime RT premix kit (iNtRON Biotechnology, Seongnam, Korea), 0.6 μl of forward primer, 0.6 μl of reverse primer, 2μl of DNA and 16.8 μl deionized sterile water).The cycle condition were the following…".

In "Antimicrobial susceptibility testing" and Fig. 1, please check

e spelling of some
antibiotics (e.g.amoxicillin instead of amoxicillin, etc).In Fig. 1, 'augmentin' is a brand name.Please, use the generic name of the drug (amoxicillin clavulanate).

In "Classification of MDR gram-negative Bacilli", the definitions of MDR are not in reference 3, as mentioned by the authors.Please, add the correct reference, Magiorakos et al., (2012) 1 .

In the sentence 'Cephalosporin resistance was def ned as resistance to ceftriaxone and ceftazidime, …', it is interesting to see that the authors did not use the optional "or" instead of "and", since resistance to these antibiotics (cephalos orins and carbapenems) can be consequence of the presence of different mechanisms.For example, Enterobacteriaceae can be resistant to ceftazidime due to the presence of ESBLs such as PER-2 or from the SHV-family, but susceptible to ceftriaxone.Same for carbapenems (e.g.deficiency of the outer membrane protein OprD confers P. aeruginosa resistance to imipenem) and aminoglycosides (different aminoglycoside modifying enzymes can confer resistance to only gentamicin or amikacin).I suggest changing these criteria, and as consequence revise the number of isolates included in this study.

In Results, "Bacterial identification", I suggest changing 'strain' by 'species' in the sentence, "Isolated Gram-negative bacilli showed different strains, including…".

The description in the second paragraph ('While isolates were distributed…' is not clear.I suggest deleting all this paragraph since Table 2 describes the same and in a very clear way.I just would keep the last 2 sentences ("K.pneumoniae was th most isolated organism from all hospital units.The distribution of different gram-negative isolates is shown in Table 2").Please, use the abbreviated form of the bacterial names after these were mentioned (i.e.first mention: Klebsiella pneumoniae, after that, K. pneumoniae; same for all the species included in this manuscript).

It is not clear the meaning of the p-values in the Tables 2 and 3. Please, clarify.

In "Antimicrobial resistance pattern of clinical isolates", Fig. 1, I don't think the authors can present accumulative results for bacterial groups that were not test d by the same antibiotics (as showed in Figure 2).I'd delete Figure 1 and the first aragraph of this section.

In "Multidrug resistance patterns among gram negative Bacilli", why were only 600 isolates were analyzed?Why were the other 134 isolates not included in this analysis?I suggest adding a sentence explaining this issue.The % of MDR (22.3%) would be an un erestimation considering my comment #4 about the criteria for defining MDR.

In Discussion, in the paragraph starting in "Resistance of gram-negative bacilli is widespread and…", the authors didn't study the mechanisms of resistance but the susceptibility of the isolates.

Then they can't talk about high rates of ESBLs.Maybe they anted to say "resistance to extendedspectrum cephalosporins" ("resistance to ESBLs" is wrong).If that is the case, the authors have to change the sentence.I suggest: "In this study, we observed high rates of resistance to extendedspectrum cephalosporins (ceftazidime and ceftriaxone), in addition to resistance to cephalexin, cefuroxime, ampicillin and amoxicillin/clavulanic acid."If there are previous studies from Sudan, and/or close countries, about AMR, it would be interesting a paragraph comparing those results with the ones obtained in this manuscript.That would show the evolution of resistance in the country/region.Please, discuss some of these data in the manuscript.


Are sufficient details of methods and analysis provided to allow replication by others? Yes

If applicable, is the statistical analysis and its interpretation appropriate?Partly 1.Is there a systematic collection of all Gram-negative bacteria isolated in the 8

s or also including outpatients?These 8 hospitals are sole hospitals in the Khartoum state,
re they representative of such state?It would be interesting to include more data on these hospitals.

2. The MDR definition adopted in the manuscript to categorize the strains as MDR is not wellreferenced.Usually, an isolate could be considered resistant to a group of antibiotics if it is resistant to at least one (≥1) antibiotic agent in the group, not necessarily to all agents in the group (as mentioned in the second paragraph of the Classification of MDR Gram negative Bacilli).I recommend the authors to revaluate their data based on a good definition as that described in the reference Magiorakos et al. (2012) 1 .

3. Reference 3 used for the definition of MDR in the introduction (and perhaps in the materials and methods) does not define such terms.Please verify that cited references matched well the text throughout the manuscript (as for reference 29 wherein the reference did not match the text).

4. Table 1, describing the bacterial gene primers used for PCR, must include a column for gene targets.In addition, it also contains many ambiguitie

to
clarify:

The primers used for Acinetobacter identification are wrongly mentioned.Indeed, the sp4F a d sp4R can identify both Acinetobacter genomic species 13TU (which is recently known as Acinetobacter nosocomialis) and A. baumannii.The primers sp2F and sp4R can identify A. baumannii.A PCR with the 3 primers (sp2F, 4F, 4R) yields 2 bands when the isolate is A. baumannii (one 294 bp with primer sp4F and sp4R and one 490 bp with sp2F and sp4R), and only one band (294 bp with sp4F and sp4R) when the isolate is A. nosocomialis.


○

For the universal 16S rRNA, the reverse primer is not present in the mentioned eference (table 1).


○

Please, also use the bona fide name for primers as they are named in their original article (for exam le instead of F use the 27F (5'-AGAGTTTGATCCTGGCTCAG-3')).11.Why are the data on drug susceptibility about Stenotrophomonas malt philia and Burkholderia cepacia and other bacteria (as Enterobacter) not mentioned in the results section and analyzed in the discussion.What are t e definitions used to define MDR in these bacterial isolates?

12. The authors define the distribution of species according to the hospital wards and specimens.However, data concerning the distribution of species according to studied hospitals is also interesting.

13. Amikacin and mipenem have a low percentage of resistance, why was amikacin not listed as the most effective antibiotic along with imipenem (paragraph one -antimicrobial resistance pattern of clinical isolates).

14.I the discussion, the authors refer the increase of resistance in their hospitals to the unrestricted use of antibiotics in Sudanese hospitals.However, the misuse of antibiotics in the community and extra-hospital set ings can also affect this upward trend.Addressing the one health concept is a way to cope with the rising threat of antimicrobial resistance.In addition, this increase in antimicrobial resistance in Sudanese hospitals may due to the occurrence of (silent) outbreaks that need investigation and control.

15. Could the author explain what did they mean in the discussion by: "In this study, we observed high rates of resistance to extended-spectrum B-lactamases (ESBL)".

16.In the discussion, the authors concluded that the resis ance rate (134 gram-negative bacilli resistant to three or four classes of antibiotics isolated over a period of 6 months) is relatively higher than the rate reported in a previous study also conducted in SUH over 30 months from January 2011 to June 2013 (reference 33).The authors of reference 33 isolated 80 bacterial strains resistant to all available antibiotics including meropenem (Microbiology records SUH).Did the authors of the manuscript isolate the same number of isolates that are resistant to all available antibiotics including meropenem as stated in the last sentence, "This concerning finding...Including meropenem")?18.The authors should be aware when formulating their findings: is actually K. pneumoniae the most commonly isolated organism from the blood specimens among all organisms?Or within Gram-negative bacteria?The same app ies to other findings?And if among all organisms, these data are not represented in the text.Please revise the text accordingly.

19.The language is generally good in the text, however, many paragraphs and sentences are written poorly.For example: "by using the following primers (Macrogen, Korea), using species-specific primers";

○ "The purity and integrity of each PCR product was evaluated electrophoresis in a 2% agarose gel"; ○ There are also many typos in the text as (use Aztreonam instead of aztreoname, settings instead of sittings, Enterobacter cloacae instead of Enterobacter colecaes) and in Figure 1 (cotrimoxazole instead of cotramoxazole, even I prefer using the same nomenclature for antibiotics in the text as well as in the figure).


○

These aforementioned examples are not exhaustive; therefore, the text should be revised accordingly, and I suggest a language edit.).For China, there are multiple systematic reviews and meta-analyses for antimicrobial resistance proportions for different pathogens available.A systematic comparison of the resistance rates described in this study with resistance patterns in other regions would help to put t

d
ta in perspective.


○

In the introduction, the authors state "This study may help in formulating antibiotic policies tailored to our hospitals.These data can be used as information for action" antibiotic stewardship and interventions to optimize antibiotic prescribing practice, therefore prolongs the usefulness of existing antibiotics.".However, in the discussion these aspects are not addressed properly.What clinical conclusions do the authors draw from their data?How do these data help to taylor ABS interventions?Etc.


○

Minor comments: I suggest a profound proof reading in order to correct spelling mistakes and typos in the manuscript.

○ I suggest adding a column "Total" in Tables 2 and 3 to indicate the total numbers of the isolated pathogens (e.g.Reviewer Expertise: infectious disease epidemiology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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243 (33.1) 230 (31.3) 183 (25) 22 (3) 26 (3.4) 30 (4.2) <0.001 *Other Gram-negative
ntimicrobial resistance pattern among different gram-negative Bacilli isolated from patients treated at Khartoum State hospitals between October 2016