Azithromycin Resistance in Clinical Isolates of Salmonella enterica Serovars Typhi and Paratyphi in Bangladesh

Abstract

Objective:

Salmonella enterica serovars Typhi and Paratyphi, the causative agents of typhoid and paratyphoid, are major threats in developing countries. The present study aimed to investigate the resistance pattern of 40 clinically isolated Salmonella enterica serovars Typhi (n = 33) and Paratyphi (n = 7) to commonly used antibiotics, particularly azithromycin.

Materials and Methods:

The disc diffusion method was used to investigate the resistance pattern of the clinical isolates against selected antibiotics. Minimum inhibitory concentration (MIC) was determined by the broth dilution method. Plate-based assays were used for the detection of efflux pumps.

Results:

It was observed that 95% of the test isolates were resistant to azithromycin and 100% were resistant to clindamycin. MIC values of azithromycin ranged between 32 and 128 μg mL⁻¹. Although 90% of isolates contained efflux pump, none of the isolates was found to have the mef(A) gene, indicating that some other efflux pump(s) might be present. Macrolide resistance gene, erm(B), was present in 25 isolates (62.5%). Other resistance genes were absent. Plasmids were absent, but class 1 integrons were present in 80% of the isolates.

Conclusions:

The occurrence of macrolide resistance in clinical Salmonella enterica serovars Typhi and Paratyphi is of particular significance in Bangladesh where azithromycin is a commonly used drug against most diseases.

Introduction

Salmonella enterica serovars Typhi and Paratyphi are responsible for enteric fever that occurs frequently in the developing world.¹ Emergence of MDR Salmonella has received special attention as this may result in treatment failure. Treatment of typhoid is rendered difficult by development of resistance to ampicillin, chloramphenicol, trimethoprim, and even to quinolones. Fluoroquinolones are more often replaced by extended-spectrum cephalosporins as a first-line treatment.^2–9 Azithromycin was proposed as a possible treatment alternative against S. Typhi isolates that show reduced fluoroquinolone susceptibility.^10,11 Salmonella isolates are intrinsically highly resistant to erythromycin, probably due to an AcrAB efflux pump.¹² However, susceptibility against azithromycin varies. In addition, azithromycin is also a safe alternative for pregnant women since quinolones are not recommended during pregnancy and pregnant women are at a higher risk of having travelers' diarrhea.¹³ Resistance to macrolides can occur owing to a variety of reasons, including the modification of target sites by enzymes such as methylases, inactivation of the antibiotic by enzymes namely esterases, phosphotransferases, and efflux with the help of pumps such as those encoded by mef and msr genes.^14–17

In Bangladesh, most diseases are treated with azithromycin, an antibiotic readily available over the counter. Earlier studies conducted in this country focused on the susceptibility of Salmonella sp. to azithromycin in terms of disc diffusion assay,^18–20 but no recent report on the determination of minimum inhibitory concentration (MIC) of the antibiotic or investigation into the resistance mechanisms to it exists. Resistance of clinical isolates of Salmonella enterica serovars Typhi and Paratyphi with increased MIC was observed in the present study. The findings of the study are of particular concern in Bangladesh where both typhoid and azithromycin are common terms to the clinicians.

Materials and Methods

Bacterial strains

A total of forty clinical samples were collected from Medinova Medical Services Ltd., a local hospital of Dhaka city, and maintained in glycerol broth at −20°C as stock cultures. Scientific and Ethics Committee approval was obtained from Dhaka University. Clinical samples isolated between June 2015 and 2016 from feces of acute Salmonellosis cases in human were identified as Salmonella enterica serovars Typhi (n = 33) and Salmonella enterica serovar Paratyphi (n = 7) by agglutination test using specific antisera (Mast, United Kingdom), biochemical tests (motility, fermentative metabolism, utilization of glucose, production of H₂S gas or other gases, and lack of oxidase), and through singleplex polymerase chain reaction (PCR) of fliC-d, prt, and tviB.^21–23 Salmonella Typhimurium ATCC 14028 was included as a reference strain for biochemical tests in this study.

Antibiotic susceptibility test

Susceptibility of test bacteria was determined against the antibiotics ceftazidime (30 μg), ceftriaxone (30 μg), clindamycin (2 μg), azithromycin (15 μg), piperacillin/tazobactam (110/10 μg), and amoxicillin/clavulanic acid (30 μg). Zone interpretation was done following established specifications.²⁴

Determination of MICs

The MIC values of azithromycin were determined following the guidelines of the Clinical Laboratory Standards Institute (CLSI, 2015).²⁴ The lowest concentration of antibiotic inhibiting visible growth of the test organisms after overnight incubation was considered MIC.

Detection of efflux pumps

Efflux pump activity was determined following the protocol described earlier.²⁵ Test bacteria were patched with sterile tips onto Mueller–Hinton agar (MHA) plates containing ethidium bromide (EtBr) at concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 μg mL⁻¹, with a numbered grid line attached on the bottom of each plate. After that the plates were covered with opaque polythene bags to avoid entry of light and were incubated at 37°C for 24 hr. Then the EtBr containing plates were observed with a UV transilluminator (Gel Doc; Bio-Rad) for fluorescence.

Effect of esomeprazole on efflux pump

Bacteria were patched with sterile cotton buds on MHA plates containing esomeprazole at concentrations of 10, 20, 50, 80, 100, 150, 200, 250, and 300 mg litre⁻¹ and incubated at 37°C for 24 hr to determine the effect of esomeprazole on the test bacteria.

Effect of efflux pump inhibition by esomeprazole on antibiotic sensitivity

Test bacteria were aseptically patched onto MHA plates containing esomeprazole (50 mg L⁻¹) and azithromycin (2, 4, 8, 16, 32, 64, and 128 μg mL⁻¹). Concurrently, the bacteria were patched onto MHA plates containing only azithromycin of the same concentrations without any esomeprazole. After 24 hr of incubation at 37°C, the plates were observed to determine the effect of esomeprazole as a proton pump inhibitor on antibiotic susceptibility of the test bacteria.

Determination of plasmid, class 1 integron, and mediator of macrolide resistance genes by PCR

Table 1 lists the primers used in this study. For singleplex PCR, 18 μL of reaction mixture (MaximoTaq Polymerase, 2X premix, GeneON, Germany) and, for multiplex PCR, 23 μL of reaction mixture were dispensed in each PCR tube. Two μL of template was then added to each tube and mixed with a pipette tip to carry out the PCR in a programmable gradient cycler (MJ Research). The PCR-amplified products and their respective sizes were determined by gel electrophoresis on 2% agarose gel. EtBr-stained gels were observed under UV transilluminator (Gel Doc; Bio-Rad). Photographs were taken and bands were analyzed with “Quality One^®” software (Bio-Rad).

Table 1.

Primers Used in This Study

Primer	Sequence (5′to 3′)	Amplicon size (bp)	Tm (°C)
int1F	AGATGTGATGGCGACGCACGA	550	55
int1R	ATTTCTGTCCTGGCTGGCGA	550	55
mphAF	GTGAGGAGGAGCTTCGCGAG	403	60
mphAR	TGCCGCAGGACTCGGAGGTC	403	60
mphBF	GATATTAAACAAGTAATCAGAATAG	492	58
mphBR	GCTCTTACTGCATCCATACG	492	58
ermAF	TCTAAAAAGCATGTAAAAGAAA	533	52
ermAR	CGATACTTTTTGTAGTCCTTC	533	52
ermBF	GAAAAAGTACTCAACCAAATA	639	45
ermBR	AATTTAAGTACCGTTACT	639	45
ermCF	TCAAAACATAATATAGATAAA	642	45
ermCR	GCTAATATTGTTTAAATCGTCAAT	642	45
ereAF	GCCGGTGCTCATGAACTTGAG	420	60
ereAR	CGACTCTATTCGATCAGAGGC	420	60
ereBF	TTGGAGATACCCAGATTGTAG	537	55
ereBR	GAGCCATAGCTTCAACGC	537	55
mefAF	AGTATCATTAATCACTAGTGC	345	55
mefAR	TTCTTCTGGTACTAAAAGTGG	345	55

Results

Antibiotic susceptibility profile

A total of 95% isolates were resistant to azithromycin and all were resistant to clindamycin (Fig. 1). The zone sizes for all isolates were less than 12 mm as indicated for resistant bacteria in the guideline (CLSI, 2015).²⁴ The isolates were more sensitive to the other antibiotics tested, particularly amoxicillin/clavulanic acid and cephalosporins.

FIG. 1.

Susceptibility of the test isolates to individual antibiotic tested. Light gray, dark gray, and black bars represent % of sensitive, intermediate, and resistant Salmonella enterica serovars Typhi (a) and Paratyphi (b), respectively.

Determination of MICs

Different test isolates of Salmonella enterica serovar Typhi showed different MICs for azithromycin. MICs for 8 isolates (20%) were found to be 32 μg mL⁻¹, whereas 21 isolates (62.5%) and 5 other isolates (17.5%) were inhibited by a higher concentration of azithromycin, 64 and 128 μg mL⁻¹, respectively. The breakpoint for MIC is 32 μg mL⁻¹ (CLSI, 2015).²⁴ On the contrary, six isolates (85.7%) of Salmonella enterica serovar Paratyphi were inhibited by 64 μg mL⁻¹ of azithromycin and MICs for the remaining isolate were found to be 128 μg mL⁻¹.

Determination of EtBr efflux mediated by Salmonella

A bacterial strain that contains efflux pump should have pumped out the lower concentration of EtBr from the cell, but at a higher saturated concentration, bacteria cannot pump out the EtBr and thereby fluoresce under the UV light. The results indicated that 81.82% (n = 33) of S. enterica serovars Typhi and 71.43% (n = 7) of serovars Paratyphi contained active efflux pump (Table 2).

Table 2.

Effect of Azithromycin on Minimum Inhibitory Concentration on Inhibition of Efflux Pump Activity

Sample no.	MIC (μg/mL)	MIC in the presence of efflux pump inhibitor	Fold of MIC inhibition	Influence of efflux pump (yes/no)
2	64	16	4	Yes
9	128	16	8	Yes
10	128	16	8	Yes
11	128	16	8	Yes
12	128	16	8	Yes
13	128	16	8	Yes
18	64	16	4	Yes
19	64	32	2	Yes
20	64	16	4	Yes
21	64	16	4	Yes
22	64	16	4	Yes
23	128	16	8	Yes
24	64	16	4	Yes
25	64	16	4	Yes
26	64	16	4	Yes
27	64	16	4	Yes
29	64	16	4	Yes
30	64	32	2	Yes
32	32	32	1	No
34	32	32	1	No
36	32	16	2	Yes
37	64	16	4	Yes
38	32	8	4	Yes
40	32	32	1	No
42	64	16	4	Yes
50	64	16	4	Yes
51	64	16	4	Yes
52	64	16	4	Yes
54	32	16	2	Yes
55	64	16	4	Yes
56	64	16	4	Yes
57	64	32	2	Yes
58	32	32	1	No
Salmonella enterica serovar Paratyphi
4	64	16	4	Yes
8	64	16	4	Yes
14	128	32	4	Yes
28	64	32	2	Yes
35	64	32	2	Yes
45	64	32	2	Yes
49	64	16	4	Yes

MIC, minimum inhibitory concentration.

Effect of efflux pump inhibitor (esomeprazole) on antibiotic resistance

The presence of efflux pump does not confirm its involvement in pumping out the antibiotic from the bacterial cell as a mechanism of drug resistance. Thus, esomeprazole and azithromycin were used in combination to check the activity of efflux pump in pumping out the antibiotic. The result showed that MIC of azithromycin significantly reduced, compared with azithromycin used alone in most of the isolates, indicating that in the presence of efflux pump inhibitor, pathogens cannot pump out the azithromycin properly and thus inhibited at a lower concentration of antibiotic and thereby confirmed its role in antibiotic resistance, as shown in Table 2.

Detection of plasmids and class 1 integron

No plasmid, neither small plasmid nor mega plasmid, was found to be present in any test isolate. However, the presence of class 1 integron was confirmed in 87.88% (n = 33) of Typhi and 42.86% (n = 7) of Paratyphi by amplification of 550 bp-sized DNA bands (Table 3). The sizes of integron bands were determined according to the 1 kb DNA marker and 100 bp DNA marker from Promega (USA) and GeneON (United Kingdom), respectively.

Table 3.

Summary of the Presence of Class 1 Integron and Erm(B) Genes in Test Isolates

Isolate	intl1	erm (B)	Isolate	intl1	erm(B)
Salmonella enterica serovar Typhi
2	+	−	30	+	+
9	+	+	32	+	+
10	+	+	34	−	−
11	+	−	36	+	−
12	+	−	37	+	−
13	+	+	38	−	+
18	+	+	50	+	+
19	+	+	51	+	+
20	+	+	52	+	−
21	+	+	54	+	+
22	+	−	55	+	−
23	+	+	56	+	+
24	+	+	57	+	+
25	+	−	58	−	+
26	+	+	40	+	−
27	+	+	42	+	−
29	−	−
Salmonella enterica serovar Paratyphi
4	+	+	35	+	+
8	−	+	45	−	+
14	+	−	49	−	−
28	−	+

Detection of mph(A), mph(B), ere(A), ere(B), erm(A), erm(B), erm(C), and mef(A) genes by PCR

Three different multiplex PCR assays were performed in this study for detecting the erm(B) and ere(A)genes, erm(A) and erm(C) genes, and mph(A), mph(B), and ere(B) genes. These genes encode the enzymes responsible for the acquisition of azithromycin resistance in bacteria. Following the PCR, only the erm(B)gene was found in a total of 25 isolates comprising 60.6% (n = 33) of Typhi and 71.4% (n = 7) of Paratyphi isolates (Table 3). However, the presence of mef(A) gene, responsible for the acquisition of efflux pump and present in both gram-positive and gram-negative bacteria, has been nullified by PCR in all isolates indicating that some other gene might be responsible for the presence of efflux pump in the test bacteria.

Discussion

It has been reported that treatment of Salmonella enterica serovars Paratyphi A infection with azithromycin failed at an MIC of 256 μg mL⁻¹ in Pakistan as early as 2010.²⁶ A similar report exists for India where MICs of ≥16 μg/mL were required to inhibit typhoidal Salmonella isolates.²⁷ As a neighboring country, Bangladesh has no similar report as yet. We found that 95% of the clinical isolates of Salmonella enterica serovars Typhi (n = 33) and Paratyphi (n = 7) were found resistant to azithromycin (95%) and 100% to clindamycin. MIC values of azithromycin against the test isolates were found to range between 32 and 128 μg mL⁻¹. According to the CLSI (2015)²⁴ guidelines, MIC values of azithromycin for resistant bacteria are ≥32 μg mL⁻¹, and hence, our isolates showed increased MICs for this antibiotic. Earlier, Gunell et al.²⁸ reported azithromycin MICs of 64–128 μg mL⁻¹ in Salmonella sp. Similarly, a Salmonella enterica serotype Stanley displayed an azithromycin MIC of 128 μg mL⁻¹.²⁹

Resistance toward multiple drugs could be for many reasons, but in the Enterobacteriaceae family it is usually associated with efflux pump(s).^30,31 In the present study, 90% of the isolates were found to have active efflux pump. To ensure that the resistance phenomena of the pathogens were due to the efflux pump, bacterial resistance toward azithromycin was tested in the presence of efflux pump inhibitor esomeprazole. It was found that in the presence of esomeprazole, a much lower concentration of azithromycin was sufficient to inhibit growth of target bacteria; the MIC values were lowered two- to eightfold for Salmonella enterica serovars Typhi and two- to fourfold for Paratyphi in the presence of esomeprazole. This finding is of interest in drug formulation where a combined therapy with a proton pump inhibitor and azithromycin can be used for inhibiting growth of Salmonella enterica serovars Typhi and Paratyphi at a lower concentration of the antibiotic.

Antibiotic resistance phenomena can spread among bacterial populations by different routes particularly through horizontal transmission via plasmids. Among the different types of plasmids, incHI1 incompatibility type appears to be particularly common in S. enterica.^32–34 However, in the present study no such plasmid was found in any clinical isolate, which was in contrast to an earlier study.^32–34 Although none of the isolates contained the plasmid, 80% of the isolates tested were found to have class 1 integron gene as determined by PCR analysis and gel electrophoresis. Thus, it might be possible that class 1 integron carries the resistance gene and spreads it to other members of the pathogens. The presence of class 1 integron in MDR Salmonella was reported.³⁵

In addition to efflux pump, encoded by mef(A) gene, macrolide resistance in Enterobacteriaceae can be mediated by several other mechanisms, including target-site modification by methylases encoded by erm genes, in particular erm(A), erm(B), and erm(C), macrolide inactivation by esterases encoded by ere(A) or ere(B) genes or phosphotransferases encoded by mph(A), mph(B), and mph(D) genes.¹⁶ There are very few reports on the mechanisms of azithromycin resistance in Salmonella. In our study, we investigated the presence of genes as mediators of macrolide resistance, namely esterases (ereA, ereB), methylases (ermA, ermB, ermC), and phosphotransferases (mphA, mphB) genes were detected by multiplex PCR. However, none of these genes was present in any isolate except ermB, which was found in 25 isolates. Although efflux pump was present in most isolates, no mef(A) gene was found indicating some other efflux pump gene may be present. Sjolund-Karlsson et al.³⁶ observed the highest MIC of 32 μg mL⁻¹ in three human isolates, which did not contain ereA, ereB, ermB, mefA, mphA, mphB, and mphD genes. The Salmonella enterica serotype Stanley was found to harbor the mphA gene²⁸ in an earlier study.

Clinical isolates of Salmonella enterica serovars Typhi and Paratyphi were found to be resistant to the macrolide group of antibiotics, including both azithromycin (95%) and clindamycin (100%). The MIC values of azithromycin were also high (32–128 μg mL⁻¹). Multiple resistance mechanisms, including efflux pump and methylase enzyme, may play a significant role in macrolide resistance. Thus, care must be taken before prescribing macrolides for Salmonella Typhi and Paratyphi infections. The dissemination of the finding of the present study to the common mass of Bangladesh is also urgent.

Footnotes

Acknowledgment

We are grateful to the University Grants Commission, Dhaka, Bangladesh, for providing research support to conduct this study.

Disclosure Statement

No competing financial interests exist.

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