β-lactamases (bla TEM, bla SHV, bla CTXM-1, bla VEB, bla OXA-1) and class C β-lactamases gene frequency in Pseudomonas aeruginosa isolated from various clinical specimens in Khartoum State, Sudan: a cross sectional study

Background: Pseudomonas aeruginosa is a pathogenic bacterium, causing nosocomial infections with intrinsic and acquired resistance mechanisms to a large group of antibiotics, including β-lactams. This study aimed to determine the susceptibility pattern to selected antibiotics and to index the first reported β-lactamases genes frequency in Ps. aeruginosa in Khartoum State, Sudan. Methods: 121 Ps. aeruginosa clinical isolates from various clinical specimens were used in this cross sectional study conducted in Khartoum State. Eighty isolates were confirmed as Ps. aeruginosa through conventional identification methods and species specific primers. The susceptibility pattern of the confirmed isolates to selected antibiotics was done following the Kirby Bauer disk diffusion method. Multiplex PCR was used for detection of seven β-lactamase genes ( blaTEM, blaSHV, blaCTXM-1, blaVEB, blaOXA-1, blaAmpC and blaDHA). Results: Of the 80 confirmed Ps. aeruginosa isolates, 8 (10%) were resistant to Imipenem while all isolates were resistant to Amoxicillin and Amoxyclav (100%). A total of 43 (54%) Ps. aeruginosa isolates were positive for blaTEM, blaSHV, blaCTXM-1, blaVEB and blaOXA-1 genes, while 27 (34%) were positive for class C β- Lactamases, and 20 (25%) were positive for both classes. Frequency of beta-lactamases genes was as follows: blaTEM, 19 (44.2%); blaSHV, 16 (37.2%); bla CTX-M1, 10 (23.3%); blaVEB, 14 (32.6%); blaOXA-1, 7 (16.3%). blaAmpC 22 (81.5%) and bla DHA 8 (29.6%). In total, 3 (11.1%) isolates were positive for both bla AmpC and blaDHA genes. Conclusion: Ps. aeruginosa isolates showed a high rate of β- lactamases production, with co-resistance to other antibiotic classes. The lowest resistance rate of Ps. aeruginosa was to Imipenem followed by Gentamicin and Ciprofloxacin. No statistically significant relationship between production of β-lactamases in Ps. aeruginosa and resistance to third generation cephalosporins was found.


Introduction
Pseudomonas aeruginosa is one of the leading causes of nosocomial infections worldwide with high mortality rates, particularly among immunocompromised patients 1 .P. aeruginosa infections are difficult to treat, due to its extraordinary antimicrobial resistance to all available classes of antimicrobial agents 2 , including β-lactams, aminoglycosides and fluoroquinolones 3 .Resistance to β-lactams occurs by different mechanisms, including bla AmpC overexpression due to genetic mutations, mutant gene acquisition, overproduction of efflux system, or low permeability 3 .β-lactamases refer to enzymes that hydrolyze the amide bond of the β-lactam ring leading to drug inactivation and therapy failure 4 .β-lactamases are classified molecularly into four groups: class A (extended spectrum β-lactamases (ESBLs)), class B (metallo-β-lactamases), class C (cephalosporinases), and class D (oxacillinases) 5 .
ESBLs are enzymes that extend their hydrolyzing ability to hydrolyze broad spectrum cephalosporins 6 and they also confer resistance to penicillins and narrow spectrum cephalosporins 3 .ESBLs are inhibited by β-lactamase inhibitors, such as clavulanic acid 7 .β-lactamases are transformed to ESBLs usually after point mutations in the β-lactamases gene.These mutations alter the substrate specificity because of changes in the amino acid sequences near the enzyme active site 5 .ESBLs producing P. aeruginosa have been reported worldwide in different countries [8][9][10][11][12][13][14][15][16][17] .
AmpC β-lactamases are class C cephalosporinases that mediate bacterial resistance to cephalosporins and cephamycins.They also exhibit low rates of monobactam, cefepime and carbapenem hydrolysis 18 and usually resist the inhibition by clavulanic acid 4 .Normally, AmpC is a chromosomal β-lactamase gene that is regulated by ampR gene and expressed constantly.Point mutations of ampR gene in Enterobacter cloacae activate AmpC that mediate resistance to β-lactams 19 .In P. aeruginosa over expressed AmpC β-lactamase mediate the resistance to broad spectrum cephalosporins 3 .AmpC β-lactamase in P. aeruginosa has also been reported in different countries around the world [20][21][22][23][24][25][26][27] .
The exact frequency of β-lactamase producing P. aeruginosa in Khartoum State, Sudan is unknown; therefore, the aim of this study was to determine the susceptibility pattern to selected antibiotics and to determine the frequency of β-lactamases producing P. areuginosa isolates collected in Khartoum State hospitals.

Study design
This is a cross-sectional study conducted between February 2017 and October 2017.Ethical approval for the study was obtained from the ethical committee of the College of Medical Laboratory Science, Sudan University of Science and Technology (SUST) (ethical meeting no, SUST/DSR/1EC/EA2/2017; data, 07 th January 2017).Written informed consent from participants was waived by the same ethical committee as the study only used previously collected human bio-specimens with limited participant data.

Collection and identification of bacterial strains
A total of 80 ps.aeruginosa clinical isolates were obtained from Soba Teaching Hospital, Elribat University Hospital, National Laboratory for Public Health, Ear Nose Throat Hospital, and Military Hospital in Khartoum State.When there was a positive confirmation of P. aeruginosa, the study supervisor went and collected the sample from the hospital.The samples were collected from patients suffering from urinary tract infections, respiratory tract infections, blood infections, and wound and ear infections.Data pertaining to the site of infection was collected from hospital records.The bacteria were preserved in 20% glycerol and peptone water and stored at -20°C Phenotypic identity of the isolates was confirmed through conventional bacterial identification methods, such as Gram stain, oxidase test, and reactions in media containing sugars, such as Kligler Iron Agar, urease test and, citrate test.Pigment production was assessed using Muller Hinton agar, and then phenotypic identity confirmed by genotypic characterization using multiplex PCR, as previously described 1 .

Antimicrobial susceptibility testing
Muller Hinton medium (HiMedia, India) was prepared and sterilized as instructed by the manufacturer.Antimicrobial susceptibility testing was performed following the modified Kirby-Bauer disc diffusion method 1 and the results interpreted according to Clinical Laboratory Standards Institute guidelines (CLSI, 2007).The following antimicrobial discs (HiMedia, India) were used for sensitivity testing: Amoxicillin (25 µg), Cefotaxime (30 µg), Amoxicillin-Clavulanic acid (30 µg), Gentamicin (10 µg), Ciprofloxacin (5 µg), Chloramphenicol (30 µg) and Imipenem (10 µg).P. aeruginosa ATCC 27853 was used as quality control strain to control the performance of the test and ensure that the test is properly performed.Phenotypic identity of the isolates was confirmed through conventional bacterial identification methods such as Gram stain, oxidase test, reactions in media containing sugars such as Kligler Iron Agar, urease test and citrate test.

Amendments from Version 2
Minor changes have been done following the reviewer's comments.Changes include specifying the final included sample size, addition of results percentages to the tables with minor edits in the figures' titles and in the abstract the Beta-lactamases genes naming changed according to the title.
Any further responses from the reviewers can be found at the end of the article

Genotypic analysis of bacterial isolates
Genomic DNA was extracted by simple boiling method 18 .The extracted DNA was used as a template for amplification of target genes using multiplex PCR using TECHNE TC-312 (UK) thermocycler.Firstly, oprI and oprL primers (Table 1) (Macrogen, Korea) were used to confirm the identification of P. aeruginosa.
Seven primer pairs (Table 1) (Macrogen, Korea) were used for detection of β-lactamases genes (bla TEM , bla SHV , bla CTXM-1 , bla VEB , bla OXA-1 ) and class C genes (bla AmpC and bla DHA ).The oprI and oprL reaction was carried out with the following cycling conditions: denaturation at 95°C for 5 min; 33 cycles of denaturation at 95°C for 30 secs, annealing at 58°C for 30 sec and extension at 72°C for 30 secs, and final extension at 72°C for 5 min 28 .
β-lactamases detection was done in two batches; the first batch was used for detection of bla TEM , bla SHV , bla CTXM-1 , bla AmpC and bla DHA , while the second batch was used for detection of bla VEB and bla OXA-1 genes.The first batch detection was done in 20 µl of final reaction mixture using Maxime PCR Premix kits (iNtRON Biotechnology, Korea), containing 13 µl of double distilled water (DDW), 0.3µl of each five forward and 0.3 µl of each five reverse primers (1.5 µl), 2 µl of Dimethyl sulfoxide (DMSO) and 2 µl of template DNA.
Amplification of the second batch was done in 20µl using Maxime PCR Premix kits (iNtRON Biotechnology, Korea), containing 14.4 µl of DDW, 0.4 µl of each two forward and 0.4 µl of each two reverse primers (0.8 µl), 2 µl of DMSO and 2 µl of template DNA.Cycling conditions for both amplification reactions were as follows: initial denaturation at 94°C for 2 minutes, then 35 cycles of denaturation at 94°C for 30 secs, annealing at 54°C for 30 secs and extension at 72°C for 50 secs, and final extension at 72° for 5 minutes.
Amplified products were analyzed by electrophoreses at 80 volts for 20 minutes on 1.5% agarose gel containing ethidium bromide and then visualized using UV transilluminator (Uvitec-UK) with 50bp or 100bp molecular DNA ladder (iNtRON Biotechnology, Korea).

Data analysis
Statistical analysis of the data was performed using chi-square test (level of significance was 0.05) with SPSS software version 20 and GraphPad prism 5 demo.
The results of antimicrobial susceptibility test for selected antibiotics are presented in Table 2.
There was a significant association between pyocyanin pigment production and site of infection (P=0.000)(Figure 1).There was also a significant association between pigment production and resistance to Chloramphenicol (P=0.020) and Cefotaxime (P=0.000), while there was insignificant association between pigment production and resistance to other antimicrobials used in this study (Figure 2).

Discussion
Pseudomonas aeruginosa is one of the main causative agents of serious nosocomial infections with increased reports of β-lactams resistant strains that makes treatment difficult and complicated 19 .Production of β-lactamases is one of the most common mechanisms of β-lactam resistance 36 .In this study the frequency of β-lactamases genes among P. aeruginosa isolates was 53.8%.This frequency is close to a report from India by Qureshi and Bhatnagar (2016) 37 , where the frequency of β-lactamases in P. aeruginosa isolates was 46%.However, the results of this study disagree with another report from India by Gupta et al. (2016) 38 where the frequency of β-lactamases in P. aeruginosa isolates was 22.9%.These discrepancies could be due to differences in strains of the clinical isolates, the antibiotics used or sample size.
The most abundant gene among the β-lactamases (bla  42 where bla VEB gene frequency was 13.3%, while the frequency of bla SHV gene was 6.6%.The report by Jamali et al. (2017) 15 concerning the genes bla TEM (15%) and bla SHV (75%) are also different from those reported in this study, and this could be due to the variation in strains of clinical isolates and the sample size used.
In this study, the frequency of class C β-lactamase genes in P. aeruginosa isolates was 27 (34%).This result is close to a report from India by Gupta et al. (2016) 38 where 43% of P. aeruginosa were AmpC producers, and disagrees with a report from Thailand by Katvoravutthichai et al. (2016) 43 where 11% of P. aeruginosa isolates were AmpC producers.In this study, out of the class C βlactamase producing P. aeruginosa isolates, 22 (81.5%) and 8 (29.6%) isolates were positive for bla AmpC and bla DHA genes, respectively.Qureshi and Bhatnagar (2016) 37 in India reported that no P. aeruginosa isolates were positive for bla AmpC gene, while Rafiee et al. (2014) 40 in Iran reported that 60.8% of P. aeruginosa were positive for bla AmpC gene.
All P. aeruginosa clinical isolates tested in our study were resistant to Amoxicillin and Amoxicillin/clavulanate and this may be due to the misuse of antibiotics in Sudan 44 , where plenty of antimicrobial agents are sold over the counter.This rate of resistance is higher than the rate of resistance reported by Ahmad et al. (2016) 45 in Pakistan where the resistance to Amoxicillin and Amoxicillin/clavulanate was 73.4% and 67.7% respectively, and this could be justified by the time difference between the studies, as well as the difference in the strains and antibiotics used.
The resistance rate of P. aeruginosa to Imipenem was 10% (n=8).This may be due to the infrequent use of Imipenem antibiotics.This percentage agrees with a study reported in Pakistan by Ahmad et     45 who found that 20.3% of P. aeruginosa isolates were resistant to Ceftazidime.The resistance rate in this study also disagrees with that reported by Albadawi (2010) 47 in Sudan who found that resistance of P. aeruginosa to Ceftazidime and Cefotaxime were 31% and 42%, respectively.These findings also indicate the rapidly increasing rates of P. aeruginosa resistance to antimicrobial agents in Sudan perhaps due to antibiotic misuse.
In this study, 23 (28.8%) and 19 (23.8%) of P. aeruginosa clinical isolates were resistant to Ciprofloxacin and Gentamicin, respectively.Altom and Ahmed (2015) 46 in Sudan also reported that 18.6% of P. aeruginosa were resistant to Gentamicin.Different results were reported by Ahmad et al. (2016) 45 , where the percentage of resistance to Gentamicin and Ciprofloxacin were 74.3% and 44%, respectively.These percentages are much higher than those reported in this study probably due to their different geographical location, study time difference and the antibiotic-use rates.
In this study, there was significant association between β-lactamases production and site of infection (P=0.030).P. aeruginosa isolated from blood showed the highest production of bla TEM , bla SHV , bla CTXM-1 , bla VEB and bla OXA-1 genes followed by ear swab, urine, sputum and wound swab.This result agrees with a study in India reported by Basak et al. (2012) 48 where the highest β-lactamases producing P. aeruginosa isolates were from blood, but disagrees with Azizi et al. (2015) 49 in Iran who found that highest production was in P. aeruginosa isolated from wound followed by urine, sputum and blood.There was insignificant association between the presence of class C β-lactamase genes and site of infection (P=0.215)found in the present study.The highest frequency of class C β-lactamases genes in P. aeruginosa were isolated from blood followed by urine, ear swab, wound swab and sputum.These results agree with the study in India reported by Basak et al. (2012)  48 in that the highest percentage of class C β-lactamase genes in P. aeruginosa were found in blood.
In the present study, there were four clinical isolates phenotypically sensitive to third generation cephalosporins (Ceftazidime and Cefotaxime) and genotypically positive for β-lactamases genes.This result indicates that P. aeruginosa may carry hidden unexpressed genes that could be detected through molecular techniques.This result agrees with a study in India reported by Bajpai et al. (2017)  50 where out of 38 phenotypically β-lactamases-negative isolates, 20 isolates were positive for β-lactamases genes.
In this study, the frequency of pigment producing P. aeruginosa isolates was 67.5%.In a study in India a higher percentage was reported where 82.5% of P. aeruginosa were pigment producers (Finlayson and Brown, 2011) 51 .The present study revealed that there was no relationship between pigment production and pattern of antimicrobial resistance, except in Chloramphenicol (P=0.02) and Cefotaxime (P=0.00).
In this study, the relationship between pigment production and site of infection was significant (P=0.000).P. aeruginosa isolated from wound infections were the highest pigment producing isolates followed by isolates from blood, urine and sputum.There was no pigment production in P. aeruginosa isolated from ear infections in this study.
The variations between the results of this study and other reports could be attributed to the difference in antibiotic usage patterns in each region, economical causes, geographical differences, sample size, differences in time in which the studies were performed, and study population.Despite the significance of the present study, there were limitations that should be avoided in future studies, such as the small sample size, phenotypic detection of β-lactamases, coverage of other β-lactamase classes, and gene sequencing should be done in order to confirm and to identify all the genes that are carried by P. aeruginosa strains in Sudan.

Conclusion
This is study is of great importance as it raises attention to the existing problem of resistance to β-lactams in P. aeruginosa in Sudan.This study confirms the reports that a number of antibiotics are becoming useless for treating this problematic bacterium, since all the strains of P. aeruginosa isolates in this study were resistant to Amoxicillin and Amoxicillin/clavulanate.The best antibiotic sensitivity results obtained in this study were those of Imipenem, followed by Gentamicin and Ciprofloxacin.Moreover, P. aeruginosa isolates showed an increased rate of β-lactamase production with co-resistance with other classes of antibiotics.Of interest is the finding that clinical isolates were resistant phenotypically in high frequencies to Amoxicillin, Amoxicillin/clavulanate and a third generation antibiotic, Cephalosporin, and showed negative results genotypically, indicating that resistance to this family of antibiotics also exist by resistance mechanisms other than β-lactamases production.Also, our PCR results revealed that P. aeruginosa possesses hidden β-lactamases genes that can't be detected phenotypically.
Finally, this study highlighted for the first time the problem of misidentification of P. aeruginosa and other microorganisms in Khartoum hospitals as only 80 out of the 120 alleged isolates were confirmed to be P. aeruginosa through PCR.

Data availability
Underlying data Figshare: SPSS, https://doi.org/10.6084/m9.figshare.12453287.v2 51is project contains the following underlying data: • SPSS.sav (Result sheet for beta-lactamases detection and sensitivity testing) • Data Dictionary.docx On the whole the research is scientifically carried out and written.Following are few minor suggestions In the abstract section, the author has mentioned that 40 isolates were other bacterial species.I think the objective of the study is the molecular detection of beta lactamases in P.aeruginosa so there is no need mention about other bacterial species.

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The statement "121 Ps. aeruginosa clinical isolates from various clinical specimens were used in this cross-sectional study conducted in Khartoum State.A total of 80 isolates were as Ps.aeruginosa through conventional identification methods and species specific primers" is really confusing.If 80 P. aeruginosa isolates were finally analyzed for drug resistance then there no need to state 121 P. aeruginosa clinical isolates.

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As mentioned above the focus of manuscript is drug resistance in P. aeruginosa, so the irrelevant information such as Pyocyanin pigment production should be deleted.

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Though in methodology section, the author has claimed statistical tests (chi square, p value), but there is no information on statistical tests in figures.I think statistical values should be included in figures.

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In all tables, please proper define the abbreviations, number and percentage (n(%)) in foot note.

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The percentage should be added in all tables, as it will help the readers to understand and interpret the tables in better way.○ Some of the references are inconsistent/incomplete which should be arranged as per journal format.

Nobumichi Kobayashi
Department of Hygiene, Sapporo Medical University School of Medicine, Sapporo, Japan The revised version is described appropriately using correct scientific terms.

Nobumichi Kobayashi
Department of Hygiene, Sapporo Medical University School of Medicine, Sapporo, Japan This is a kind of primitive study that provides only limited information.Only importance is that this study was done in Sudan.However, still number of isolates were low, and errors in descriptions of technical terms are found in this manuscript.
Abstract: Number of P. aeruginosa should be written as "80"."121" should not appear here, because 41 isolates were not P. aeruginosa. 1.
Amoxyclav may be a commercial name of the drug.It should be written as general name.Maybe it is "amoxicillin/clavulanate".

3.
Authors may have serious misunderstanding regarding the term of ESBL.NOT all of TEM, SHV, OXA type beta-lactamases are ESBL.TEM-1, 2, SHV-1, OXA-1 are not ESBL.Other subtypes of TEM, SHV, and OXA are called as "ESBL".Those subtypes can be identified only by sequencing of full-length ORF of these genes.In this manuscript, only CTX-M type gene can be called as "ESBL".However, it is not sure that TEM, SHV, OXA type genes are ESBL genes or not, because sequence was not determined.In this regard, this manuscript must be revised substantially.Authors can mention CTX-M as ESBL, others should not be referred as ESBL, but just individual enzyme nams, e.g., "TEM gene".According to this alteration, whole manuscript should be rewitten.

4.
CTX-M-1 should be written as "CTX-M-1 group".CTX-M types cannot be determined by only PCR.Authors should read and study literature on ESBL and betalactamases of bacteria, to exactly understand their nomenclature and definition.

5.
Table 3 cannot be understood.Authors should make a table to show combination pattern of beta-lactamases and their frequency.

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 Are all the source data underlying the results available to ensure full reproducibility?
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Figure 1 .
Figure 1.Frequency of Pseudomonas aeruginosa pigment producing isolates according to site of infection.

Figure 2 .
Figure 2. Frequency of Pseudomonas aeruginosa pigment producing isolates according to antimicrobial resistance.

Figure 4 .
Figure 4. Frequency of class C genes among Pseudomonas aeruginosa isolates and site of infection.

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Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.Reviewer Expertise: Medical MicrobiologyI 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.©2020 Kobayashi N.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1 Reviewer
Are all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.Reviewer Expertise: microbiology, public health 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.VersionReport 26 August 2020 https://doi.org/10.5256/f1000research.27382.r68073© 2020 Kobayashi N.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.