Incidence of Oxa23 and Oxa51 Genes Associated with Bacterial Isolated from Patients with Urosepsis: Single Centre Prespective

Background: Urosepsis is one of the most common infections that require empirical broad spectrum antibiotics immediately after diagnosis. This has led to development of bacterial resistance by acquiring the capability to destroy the β-lactam ring. Methodology: This is a cross-sectional hospital-based study. The study was conducted from 2019 to 2020 at Gezira Hospital for Renal diseases and surgery (GHRDS). A hundred patients were diagnosed clinically with urosepsis and the isolated organisms were Escherichia coli, Staphylococcus aureus, Proteus mirabilis, Klebsiella pneumonia and Pseudomonas aeruginosa. The susceptibility test was conducted by Kirby Bauer disc diffusion technique according to clinical laboratory standard institute (CLSI) guidelines. Seventy eight samples of bacterial genomic DNA were confirmed by 16srRNA and multiplex PCR, were performed for genotypic blaOXA-51 and blaOXA-23 gene characterization of isolated bacteria. Then gel electrophoresis was used to identify the presence or absence of (blaOXA-51 and blaOXA-23) genes. Results: 88.5% (69/78) in 16srRNA detected. Using multiplex PCR, the frequencies of blaOXA-51 and blaOXA-23 genes were 13% and 10.1%, respectively. The percentages of isolates which yielded both blaOXA-51 and blaOXA-23 among P. aeruginosa was 25% (1/4), among K. pneumonia was 17% (1/6), and among E. coli was 8% (3/37). Only blaOXA-51 was detected in P. mirabilis 10% (1/10) and only blaOXA-23 was detected in S. aureus 5% (1/18). Conclusion: In this study, the presence of blaOXA-51 and blaOXA-23 genes was increased in the isolated bacteria.

• Urosepsis
• Carbapenem-Resistant Enterobacteriaceae (CRE)
• blaOXA-51 and blaOXA-23

1. Introduction

The most common isolated pathogens that cause urosepsis are Escherichia coli, followed by Proteus mirabilis, Enterobacter, Klebsiella pneumonia, Pseudomonas aeruginosa, and gram-positive bacteria [3] [4] [5]. These causative organisms are considered serious invaders which need urgent treatment and are classified into critical (including; P. aeruginosa, E. coli, P. mirabilis, K. pneumonia) and high (including; S. aureus) according to World Health Organization (WHO) [6]. The customary empirical usage of late-generation of cephalosporins yields unlimited development of bacterial resistance [7]. The majority of these strains acquired the capability to destroy the β-lactam ring [8] [9] [10] in cephalosporins. Therefore carbapenems were used as an effective alternative to the broad-spectrum antibiotic to treat these bacteria. However, organisms express resistance to carbapenems [11].

Globally; in the last decade strains that revealed resistance to carbapenemfell mainly in gram negative bacteria including; Acinetobacter baumannii and Pseudomonas aeruginosa by different mechanisms intrinsic or mediated by transferable carbapenemase-encoding genes [12] these mechanism includes; decreased membrane permeability, efflux pumps and an enzymatic resistance to carbapenems. Carbapenemase is enzymatically classified into Ambler class A, B and D based on amino acid homology [13] [14] [15]. Acquired class D β-lactamases, also known as oxacillin hydrolyzing enzymes (OXA) genes, structurally are different in amino acid from class A and C enzymes and are widely distributed among gram negatives rods. This resistance is mostly associated with class 1 integron or insertion sequences. These genes are characterized by important genetic diversity and great heterogeneity in terms of β-lactam hydrolysis spectrum [16]. This class contains various types of carbapenemase and generally cannot be deactivated by clavulanic acid, tazobactam, and sulbactam and it is including blaOXA-51, blaOXA-23, blaOXA-24/40, blaOXA-58, blaOXA-143, and blaOXA-235. Most of them are encoded by chromosomal genes with OXA-48 considered the most commonly detected [15] [17] [18].

The recent acquisition and encoding of carbapenem-resistance by chromosomal genes (OXA-51), plasmid-mediated genes (OXA-23) [11] [19] [20], and production of cephalosporinases combined with mutations associated with decreased permeability of the bacterial cell wall. These enzymes have the capability to hydrolyze and inactivate beta lactam rings are found in beta lactam antimicrobials [17] [21], which led to emergence of carbapenem-resistant enterobacteriaceae (CRE) [22] [23].

Carbapenem resistance (CR) is globally distributed in most bacteria [24] [25]. In Sudan many studies dealt with extended-spectrum beta-lactamases (ESBL) genes and carbapenem resistant [26] [27]. This study aims to determine the incidence of oxa23 and oxa51 among isolated bacteria from urosepsis patients admitted to Gezira Hospital for Renal Diseases and Surgery, Sudan (GHRDS).

2. Methodology

2.1. Patients and Methods

This is a cross-sectional hospital-based study. It was conducted from 2019-2020 at (GHRDS), Wad Medani, Sudan. GHRDS is considered the only specialized referred centre outside Khartoum. A hundred samples were selected from patients diagnosed clinically in urology department with urosepsis caused by drug-resistant pathogens. The participants were in different ages and genders and came from different States in Sudan.

2.2. Identification of Bacterial Isolates and Susceptibility Testing

Urine samples were collected in sterile urine containers then inoculated on cysteine lactose electrolyte deficient (CLED) media, overnight at 37˚C, then identified by colonial morphology, lactose fermentation and gram’s stain. Further biochemical tests identified the bacterial isolates and the antimicrobial susceptibility testing was performed by Kirby Bauer disc diffusion technique for selected antimicrobial agent according to clinical and laboratory standards institute (CLSI). The most common β-lactam antibiotics prescriptions in policy of urology department of GHRDS are third and fourth cephalosporin (ceftriaxone, ceftizoxime and cefepime) and carbapenem (meropenem).

2.3. Molecular Identification

2.3.1. Bacterial Genomic DNA Extraction

Extracted genomic DNA fromisolated bacteriain different antimicrobial susceptibility pattern were performed using (G-spin TM Total) iNtron (South Korea, Soul), Lot. No. 105251551. The extracted DNA was stored at −80˚C till PCR analysis.

2.3.2. 16srRNA Amplification

From extract used Primers for 16srRNA amplification, blaOXA-51 and blaOXA-23 showed in Table 1. Measurement of DNA concentration and purity quality was accomplished with a NanoDrop spectrophotometer (Bibby Scientific, UK).

2.3.3. PCR Protocol Used for Multiplex to Detect blaOXA-51, blaOXA-23

In this study, we performed two molecular tests. A single PCR reaction for the detection of bacterial 16srRNA gene and a multiple exprotocol for detection of OXA-23 and OXA-51 genes In the protocol of 16srRNA, the initial denaturation was done at 95˚C for 6 minutes for initial denaturation followed by 40 cycles at 95˚C for 30 seconds for denaturation, 50˚C for 50 seconds for annealing, and 72˚C for 60 seconds for extension. The final extension was 10 minutes at 72˚C. In the protocol of multiplex PCR, an initial denaturation at 94˚C for 3 minutes is followed by 35 cycles of 94˚C for 45 seconds, 57˚C for 45 seconds, and 72˚C for 1 60 seconds, with a final extension for 5 minutes at 72˚C.

The PCR product processed in 1% agarose gel electrophoresis was performed to identify the presence or absence of (blaOXA-51 and blaOXA-23) genes.

3. Ethical Approval

The ethical approval was obtained from GHRDS, Faculty of Medical Laboratory Sciences, University of Gezira and Ministry of Health, Gezira State, Sudan.

4. Results

From 100 patients diagnosed clinically as urosepsis in the urology department of GHRDS during the study period, the majority of samples were males from different states with a median age of 45years. The result was shown in (Table 2). Urine samples were cultured on CLED aerobically along with urinalysis. E. coli was the predominant bacteria with a high frequency of isolation (Figure 1). Antimicrobial susceptibility test (AST) showed most of the isolates were prevalent with complete resistance to cefepime as shown in (Table 3). The identities of 88.50% (69/78) of isolates were confirmed with 16srRNA. The length of bands was approximately 1500 bp as shown in (Figure 2). 16srRNA of E. coli was detected at 89.2% (33/37), with that of S. aureus at 85.7% (18/21), P. mirabilis at 10/10 (100%), K. pneumonia at 83.3% (5/6), and P. aeruginosa at 25% (1/4) as

Table 1

Primers use for amplification of 16srRNA, oxa23 and oxa51 in this study

Primer	Sequence	Fragment size
16srRNAF 16s RNA R	5’-AGAGTTTGATCCTGGCTCAG-3’ 5’-GGTTACCTTGTTACGACTT-3’	1500	[28]
Oxa23 F Oxa23 R	5-GAT CGG ATT GGAGAACCA GA-3 5-ATT TCTGACCGC ATT TCC AT-3	501 bp	[29]
Oxa51 F Oxa51 R	5-TAATGCTTT GAT CGGCCT TG-3 5-TGG ATT GCACTT CAT CTTGG-3	353 bp	[29]

Table 2

Social demographic data of urosepsispatients in GHRDS 2019-2020

		Patients (n)	Percent
Gender	Male	70	70
	Female	30	30
	Total	100	100
Age	1 - 16 Years	7	7
	17 - 40 Years	18	18
	41 - 60 Years	31	31
	>61 Years	44	44
	Total	100	100
State	Gezira	69	69
	Sinnar	11	11
	Elqadarif	7	7
	Eldamazin	10	10
	Others	3	3
	Total	100	100

Table 3

The results of antimicrobial susceptibility/intermediate-resistant/resistant of isolated strains

	Sensitive		Intermediate		Resistant
	F	%	F	%	F	%
Meropenem	70	90	0	0	8	10
Ceftizoxime	42	54	9	12	27	35
Ceftriaxone	11	14	7	9	60	77
Cefepime	0	0	0	0	78	100

showed in Table 4. Obtained bands by multiplex PCR for OXA-23 and OXA-51 were 501 bp and 353 bp, respectively (Figure 3). The frequency of blaOXA-23 and blaOXA-51 gene 10.1% and 13%, respectively showed in Table 5. Presence of

Table 4

Detected DNA of isolated bacteria by 16srRNA

Isolated Microorganism	No Product		Detect		Total
	F	%	F	%
E. coli	4	11	33	89	37
S. aureus	3	14	18	86	21
P. mirabilis	0	0	10	100	10
K. pneumonia	1	17	5	83	6
P. aeruginosa	3	75	1	25	4
Total	11	14	67	86	78

Table 5

The incidence of blaOXA-51 and blaOXA-23

	Oxa51		Oxa23
	Frequency	Percent	Frequency	Percent
Absent	60	87	62	89.9
Present	9	13	7	10.1
Total	69	100	69	100

blaOXA-51 and blaOXA-23 among isolated resistant strain associated with result of susceptibilty, cefepime showed full resistance, furthermore; meropenem and ceftizoxime showed significant p value 0.000, shown in Table 6 & Table 7. Eight percent (3/37) and 14% (3/18) of E. coli and S. aureus isolates possessed blaOXA-51 gene. While 25% (1/4) of P. aeruginosa showed blaOXA-51 and blaOXA-23 genes (Table 8).

Table 6

The correlation between the presence of oxa23 and antibiotics resistance

Antibiotics	Susceptibility	Oxa23				P value
		Absent		Present
		F	%	F	%
MRP	Sensitive	57	100	0	0	0.000
	Resistant	5	42	7	58
	Total	62	90	7	10
CZX	Sensitive	39	100	0	0	0.000
	Resistant	17	71	7	29
	Intermediate	6	100	0	0
	Total	62	90	7	10
CRO	Sensitive	10	100	0	0	0.262
	Resistant	46	87	7	13
	Intermediate	6	100	0	0
	Total	62	90	7	10
CPM	Resistant	62	90	7	10	NA
	Total	62	90	7	10

MRP: Meropenem; CZX: Ceftizoxime; CPM: Cefepime; CRO: Ceftriaxone.

Table 7

The correlation between the presence of oxa51 and antibiotics resistance

Antibiotics	Susceptibility	oxa51				P. value
		Absent		Present
		F	%	F	%
MRP	Sensitive	57	100	0	0	0.000
	Resistant	3	25	9	75
	Total	60	87	9	13
CZX	Sensitive	39	100	0	0	0.002
	Resistant	15	63	9	37
	Intermediate	6	100	0	0
	Total	60	87	9	13
CRO	Sensitive	10	100	0	0	0.388
	Resistant	44	83	9	17
	Intermediate	6	100	0	0
	Total	60	87	9	13
CPM	Resistant	60	87	9	13	NA
	Total	60	87	9	13

MRP: Meropenem; CZX: Ceftizoxime; CPM:Cefepime; CRO: Ceftriaxone.

Table 8

Presence of blaOXA-51 and blaOXA-23 in bacterial isolates

Isolated Microorganism	Oxa51				Oxa23				Total
	Absent		Present		Absent		Present
	F	%	F	%	F	%	F	%
E. coli	34	92	3	8	34	92	3	8	37
S. aureus	18	86	3	14	20	95	1	5	21
P. mirabilis	9	90	1	10	10	100	0	0	10
K. pneumonia	5	83	1	17	5	83	1	17	6
P. aeruginosa	3	75	1	25	3	75	1	25	4
Total	69	88	9	12	71	91	7	9	78

5. Discussion

Urosepsis is a sepsis condition resulting from a dysregulated response to infections of urinary tract and/or male genital tract, its either community-acquired or healthcare associated infections remain in hospital, and needs medical intervention from starting antimicrobial therapy to the causes of the obstructive uropathy [4] this intervention was important risk factors of acquiring carbapenem resistant bacteria [15] [30]. This study highlights the incidence of carbapenem resistant genes, among E. coli, K. pneumonia, P. mirabilis, P. Aeruginosa and S. aureus isolated from urosepsis patients.

In this study males were more affected than females with average age 45 years old, because this study conducted in urology units at GHRDS and most admitted of patients suffering prostate cancer, benign prostatic hyperplasia (BPH) and urethral stricture, this result was agreed with study carried out by Ibrahim et al., Goveas and Muhammad et al. study [31] [32] [33].

The isolates showed phenotypically high resistant rate to different groups of antibiotics this result similar to Ibrahim et al., Ehssan studied in Sudan and Mohamed et al. studied in Cameroon, observed by using disc diffusion methods [31] [34] [35]. Globally, the outbreak of resistance to most prescribed antibiotics especially in complicated UTIs (cUTIs), urosepsis and pyelonephritis [36] [37], its led to exchange of mutant genes by intrinsic by transferable carbapenemase-encoding genes [12].

Most of these organisms observed contain ESBL genes [38], because carbapenem is the best option drug used to treat it [10]. Unfortunately, this study didn’t test forESBL genes about isolated bacteria, but Ibrahim et al. reported 45.1% from isolated bacteria containing ESBL genes in Khartoum teaching hospital, Sudan [26].

In this study the rate of CR phenotypically was 45.3%, this result is inagreement with CLSI and European committee on antimicrobial susceptibility testing standards were 17.4% and 10.9%, respectively [24]. The highest percentage of CR in this study as a result of being limited in β-lactam antibiotics used in urology department. According to MDR phenotypes, this study observes same frequency of isolated resistance among Latin America (41.1%) [24]. The detection of oxa23 and oxa51 in this was confirmed to the phenotypic resistance to carbapenem groups from isolated bacteria. Moreover, the detected genes observed in resistant strains to meropenem and was highly significant p value (p = 0.000).

The study finding the incidence of chromosomal and genes plasmid-mediated genes (blaOXA-51, blaOXA-23) were reported 13% and 10.1 respectively, it agrees with Mohamed et al. studied in the predominate gene but differs in the incidence, while the rate of blaOXA-51 gene and blaOXA-23 27.8% (10/36) and 2.7% (1/36), respectively [27].

Recently, some strains such as P. Aeruginosa, K. pneumonia, and E. coli appear the chromosomal genes blaOXA-51 and acquired blaOXA-23 gene among CRE these genes that appear only in Acinetobacter species [17] [27]. In this study CR gene (blaOXA-51, blaOXA-23) were found in P. aeruginosa 25% (1/4), K. pneumonia 17% (1/6), S. aureus 14% (3/18), E. coli 8% (3/37), P. mirabilis 10% (1/10) detected only in blaOXA-51 and the frequency of S. aureus in blaOXA-23 was 5% (1/18), although E. coli the predominant isolated, it has the lowest incidence in appearance of chromosomal genes blaOXA-51 among CRE [18] [23]. On the other hand, observed there is no study about these genes among S. aureus. Sample size played major role in the variation of the incidence and percentage between this study and Mohamed et al. study [27].

6. Conclusion

This study has shown a high rate of CR (blaOXA-23 and blaOXA-51) genes among the isolated bacteria collected from GHRDS, the E. coli was the predominant bacteria with the least frequency of blaOXA-23 and blaOXA-51 presence genes than other isolates. This result needs to focus on the higher frequency bacteria to CR gene especially P. aeruginosa and how to treat them without ignoring E. coli.

Study Limitation

This study is limited to imipenem and ertapenem as groups of carbapenem and conducted only in meropenem with third and fourth cephalosporin, and limited to ESBL genes in the detections.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

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