Prevalence of gyrA Mutations in Nalidixic Acid-Resistant Strains of Salmonella Enteritidis Isolated from Humans,Food,Chickens,and the Farm Environment in Brazil

Abstract

Salmonella Enteritidis strains that are resistant to nalidixic acid and exhibit reduced susceptibility to fluoroquinolones have been increasing worldwide. In Brazil, few studies have been conducted to elucidate the quinolone resistance mechanisms of S. Enteritidis strains. This study analyzed the profile of gyrA, gyrB, parC, and parE mutations and plasmid-mediated quinolone resistance (PMQR) mechanisms in S. Enteritidis Nal^R strains isolated in Brazil. Moreover, the minimum inhibitory concentrations (MICs) of ciprofloxacin were evaluated in 84 Nal^R strains and compared with 20 Nal^S strains. The mutation profiles of the gyrA gene were accessed by high-resolution melting analysis and gyrB, parC, and parE by quinolone resistance-determining region sequencing. The MICs of ciprofloxacin were accessed with Etest^®. The strains were divided into five gyrA melting profiles. The Nal^R strains exhibited the following amino acid substitutions: Ser97→Pro, Ser83→Phe, Asp87→Asn, or Asp87→Tyr. The average MICs of ciprofloxacin was 0.006 μg/ml in the Nal^S and 0.09 μg/ml in the Nal^R strains. No points of mutation were observed in the genes gyrB, parC, and parE. The qnrB gene was found in two strains. In conclusion, the reduced susceptibility to ciprofloxacin observed in Nal^R strains may cause treatment failures once this drug is commonly used to treat Salmonella infections. Moreover, this reduced susceptibility in these Brazilian strains was provided by target alteration of gene gyrA and not by mobile elements, such as resistance plasmids.

Introduction

Gastroenteritis due to Salmonella is a major health problem worldwide that is linked to the consumption of contaminated food. Among the serovars of Salmonella enterica, the Enteritidis (S. Enteritidis) has remained the most frequently isolated in gastroenteritis cases in many countries since the 1980s.^1–3

In the United States, Salmonella is estimated to cause every year 1 million foodborne illnesses with 19,000 hospitalizations and 380 deaths. Recent reports show that in 2014 serovar Enteritidis was the most isolated, representing 19% of the Salmonella isolates in that country.⁴ In European countries, among the 82,694 confirmed salmonellosis cases in 2013, serovar Enteritidis was the most isolated, representing 39.5% of the reports.⁵

The use of antimicrobials is not recommended in cases of gastroenteritis, which are usually self-limiting. However, in some cases, the infection might lead to serious infections, and the antibiotic therapy might be necessary.^6,7

Nalidixic acid was the first quinolone and was widely used in the early 1960s to treat Gram-negative bacterial urinary tract infections. Further modifications in the chemical structure of these drugs, such as the addition of a fluorine atom, increased the efficacy of this group.^6,8–11 The drug of choice for the treatment of Salmonella infections is typically ciprofloxacin due to its broad-spectrum antimicrobial activity. In the veterinary field, fluoroquinolones have been used for the treatment of infections or as growth promoters in food production animals.^7,12

The extensive use of these antimicrobials has led to increasing numbers of non-typhi Salmonella strains that are resistant to quinolones and exhibited reduced susceptibility to fluoroquinolones.^10,13–19 This reduced susceptibility can lead to treatment failures in some cases.^13,15

It has been reported that quinolone resistance among Salmonella serovars has been increasing over the years in several countries, such as the United States, European countries, Southeast Asia, among others.²⁰ In the United States, around 3% of all non-typhoidal Salmonella isolated between 2009 and 2011 presented some level of resistance to ciprofloxacin.²¹

Quinolones target the bacterial DNA gyrase; this enzyme is a type II topoisomerase that is essential for bacterial DNA replication.²² This enzyme consists of 2A and 2B subunits encoded by gyrA and gyrB genes, respectively. The second target of the fluoroquinolones is the topoisomerase IV enzyme, which is codified by the genes parC and parE.^7,8,20,22

The mechanisms linked to quinolone resistance in Salmonella include changes in the binding site of the antimicrobial to the enzyme. These changes are related to mutations in the quinolone resistance-determining region (QRDR) of the target genes. These points of mutation can lead to amino acid substitutions that result in different levels of quinolone resistance.^7,19,23,24

Although four target genes exist, gyrA is the main gene involved in quinolone resistance, and a single point of mutation in the QRDR of this gene typically results in resistance to nalidixic acid and reduced susceptibility to fluoroquinolones. Two or more mutations in the gyrA gene or in the other topoisomerase genes confer resistance to ciprofloxacin. The QRDR of DNA gyrase A in S. enterica is localized between amino acids 67 and 122.^23,25

Other resistance mechanisms include the qepA, oqxA, and oqxB genes that codes an efflux pump that does not permit the intracellular accumulation of the antimicrobial agent, the actions of the plasmid gene qnr that codes a group of pentapeptide proteins that bind to DNA gyrase and prevent the action of quinolones, and the action of the aminoglycoside acetyltransferase that is coded by gene aac(6′)-Ib-cr and can reduce susceptibility to ciprofloxacin.^{7,19,22,26–28}

Recent studies from our research group regarding S. Enteritidis strains isolated from humans, food, and chickens in Brazil revealed a high level of nalidixic acid resistance, particularly in poultry-related strains.^29,30 However, the levels of resistance to fluoroquinolones and the mechanisms of quinolone resistance were not evaluated in those studies. In Brazil, the only study of quinolone resistance mechanisms was conducted with Salmonella strains from several serovars that were isolated only in the Paraná State over the period of 8 years from 1999 to 2007.³¹

Thus, the objective of this study was to analyze the profile of gyrA mutations and investigate the presence of some plasmid-mediated quinolone resistance (PMQR) genes that are related to quinolone resistance, such as qnr genes, qepA, and aac(6′)-Ib-cr, in nalidixic acid-resistant (Nal^R) S. Enteritidis strains isolated in Brazil. The studied strains were isolated from humans, food, chickens, and farm environments in five Brazilian States in four regions over a 44-year period. Moreover, the minimum inhibitory concentrations (MICs) of ciprofloxacin for Nal^R S. Enteritidis strains were evaluated and compared with those of nalidixic acid-susceptible strains (Nal^S). These data were analyzed to evaluate the prevalent points of mutation in the gyrA gene, and to verify the presence of other mechanisms of quinolone resistance in S. Enteritidis strains from Brazil.

Materials and Methods

Bacterial strains

A total of 84 Nal^R Salmonella Enteritidis strains were studied (Table 1). These strains were composed of the nalidixic acid-resistant strains that were identified among 188 S. Enteritidis strains that were studied in previous works by our research group.^29,30 Additionally, four Nal^R strains isolated in 1969, 2012, and 2013 were included in this study (Table 1), which were identified among 26 new strains received by our laboratory and isolated between 1968 and 1993 and in 2012 and 2013 (unpublished data). The strains were isolated from human feces (24), food (16), chickens (29), and farm environments (15) (Table 1). Moreover, 20 nalidixic acid-sensitive (Nal^S) S. Enteritidis strains that were representative of the four sources and years of isolation were used for comparisons with the resistant strains (Table 1). Those strains were provided by two Salmonella spp. References Laboratories in Brazil that were the Adolfo Lutz Institute of Ribeirao Preto (IAL-RP) and Oswaldo Cruz Foundation (FIOCRUZ) and by AVIPA (Avicultura Integral e Patologia S/A).

Table 1.

Year, Source, Region, and State of the 84 Salmonella Enteritidis Nalidixic Acid-Resistant Strains Studied and Isolated in Brazil

Total of strains^a	Year of isolation	Source	Region of isolation	States of Brazil
1	1969	Human	Southeast	Rio de Janeiro
1	1996	Human	Southeast	São Paulo
3	1999	Food^b	Southeast	São Paulo
5	2000	Human/food	Southeast	São Paulo
2	2001	Human/food	Southeast	São Paulo
2	2002	Human/food	Southeast	São Paulo
3	2003	Food	Southeast	São Paulo
12	2004	Human/food/chickens/farm environment	Southeast/Midwest/Northeast/South	São Paulo/Mato Grosso/Pernambuco/Paraná
6	2005	Chickens	Southeast/Northeast/South	São Paulo/Pernambuco/Paraná
9	2006	Human/chickens/farm environment	Southeast/Northeast	São Paulo/Pernambuco
7	2007	Chickens/farm environment	Southeast/Northeast/South	São Paulo/Pernambuco/Paraná
9	2008	Chickens/farm environment	Southeast/Northeast	São Paulo/Pernambuco
14	2009	Human/chickens/farm environment	Southeast	São Paulo/Pernambuco/Minas Gerais
7	2010	Human/food/chickens/farm environment	Southeast/Northeast	São Paulo/Pernambuco
1	2012	Human	Southeast	Rio de Janeiro
2	2013	Human/food	Southeast	Rio de Janeiro
Total = 84

The 26 strains with reduced susceptibility to ciprofloxacin presented MICs between 0.12 and 0.5 μg/ml and were isolated from the four sources in the States of São Paulo, Mato Grosso, Pernambuco, and Paraná in 1996, 1999, 2000, 2001, 2004, 2005, 2006, 2008, 2009, and 2010.

Strains isolated from food include mayonnaise, sandwich, cake, pork, raw chicken, papaya, raw fish, and beef.

MICs, minimum inhibitory concentrations.

Detection of mutations in the QRDR of gyrA, gyrB, parC, and parE

Mutations in the QRDR of the gyrA gene were searched in the 84 Nal^R and 20 Nal^S strains of S. Enteritidis (Table 1). Initially, the different mutation profiles were assessed through high-resolution melting analysis (HRMA) using the general procedure described by Souza and Falcão³² with a few modifications. The primers used were identical to those presented by Slinger et al.³³ and amplified a 89-bp fragment of the QRDR in the gyrA gene of S. enterica (Table 2).

Table 2.

Sequences, Amplicon Sizes, and References for the Primers Used in the Present Study

Primers		Sequences (5′–3′)	Amplicon size (bp)	References
SA89 (HRMA)	Forward	GAC GTA ATC GGT AAA TAC CAT CC	89	Slinger et al.³³
	Reverse	ATG TAA CGC AGC GAG AAT G
gyrA1	Forward	CAT GAA CGT ATT GGG CAA TG	305	Jeong et al.¹⁹
stgyrA2	Reverse	CCG TAC CGT CAT AGT TAT CC
SgyrB.1	Forward	GAA TAC CTG CTG GAA AAC CCA T	446	Fàbrega et al.³⁷
SgyrB.2	Reverse	CGG ATG TGC GAG CCG TCG ACG TCC GC
parC.Sal1	Forward	AAG CCG GTA CAG CGC CGC ATC	395	Fàbrega et al.³⁷
parC.Sal2	Reverse	GTG GTG CCG TTC AGC AGG
SparE.1	Forward	CCT GCG GCC CGG CGT TGC CGG GG	465	Fàbrega et al.³⁷
SparE.2	Reverse	CGC CCG CCT TCT CTT CTT CCG TCA GCG CG
aac(6′)-Ib-cr	Forward	TTG CGA TGC TCT ATG AGT GGC TA	482	Park et al.³⁹
	Reverse	CTC GAA TGC CTG GCG TGT TT
qnrA	Forward	ATT TCT CAC GCC AGG ATT TG	574	Jacoby et al.⁴⁰
	Reverse	TGC CAG GCA CAG ATC TTG AC
qnrB	Forward	CGA CCT KAG CGG CAC TGA AT	513	Jacoby et al.⁴⁰
	Reverse	GAG CAA CGA YGC CTG GTA GYT G
qnrC	Forward	GGG TTG TAC ATT TAT TGA ATC G	307	Kim et al.⁴¹
	Reverse	CAC CTA CCC ATT TAT TTT CA
qnrD	Forward	TGT GAT TTT TTC AGG GGT TGA	520	Tamang et al.¹⁰
	Reverse	CCT GCT CTC CAT CCA ACT TC
qnrS	Forward	ACT GCA AGT TCA TTG AAC AG	431	Jacoby et al.⁴⁰
	Reverse	GAT CTA AAC CGT CGA GTT CG
qepA	Forward	AAC TGC TTG AGC CCG TAG AT	596	Kim et al.⁴¹
	Reverse	GTC TAC GCC ATG GAC CTC AC
oqxA	Forward	GAC AGC GTC GCA CAG AAT G	339	Chen et al.⁴²
	Reverse	GGA GAC GAG GTT GGT ATG GA
oqxB	Forward	CGA AGA AAG ACC TCC CTA CCC	240	Chen et al.⁴²
	Reverse	CGC CGC CAA TGA GAT ACA

HRMA, high-resolution melting analysis.

Briefly, HRMA is considered to be a fast, cost-effective, sensitive, specific, and high-throughput method for the interrogation of single-nucleotide polymorphisms (SNPs), hypervariable repeat regions in polymerase chain reaction (PCR) products, and also for the discovery of new SNPs.³⁴ It consists of a one-step, closed-tube, post-PCR assay that detects sequence variations within specific loci through accurate monitoring of the reduction in the fluorescence of a PCR product stained with a double-stranded specific fluorescent dye to monitor the transition from unmelted to melted DNA. In contrast to traditional melting analysis, the information of the HRMA method is contained in the shape of the melting curve, rather than just in the calculated melting temperature.³⁵

Heteroduplexes were generated to separate the profiles that exhibited mutation points with complementary bases (e.g., A-T and T-A) through the addition of an exogenous DNA sample with a known sequence.

Representative strains of each group generated by HRMA were sequenced to analyze the type of mutation in each profile. Moreover, representative strains of each group comprising 20 Nal^R and 10 Nal^S strains had their genes gyrB, parC, and parE sequenced to check possible mutation points in the QRDR of these genes. The general procedure for sequencing was the same as that described by Souza et al.³⁶ and used the primers gyrA1, stgyrA2, SgyrB.1, SgyrB.2, parC.Sal, parC.Sal2, SparE.1, and SparE.2 (Table 2).^19,33,37 Each forward and reverse strand was sequenced at least two times.

Sequence analysis

The resulting DNA sequence of the QRDR of each strain described in the previous section was checked for quality by visual inspection with the aid of the ChromasPro version 2.33 software (Technelysium Pty. Ltd.) and trimmed to remove the low-quality nucleotide sequences from the ends. This process generated sequences with the same number of nucleotides that were consequently of the same size.

Detection of PMQR genes

The genomic DNA of the 84 studied strains (Table 1) was extracted as described by Campioni and Falcão.³⁸ The primers used in the reaction, the annealing temperatures, and the references for the primers used are described in Table 2.^10,39–42 The genes under study were the qnrA, qnrB, qnrC, qnrD, and qnrS genes, which are code for a group of pentapeptide proteins, the qepA, oqxA, and oqxB, which are code for efflux pumps, and the aac(6′)-Ib-cr, which codes for an aminoglycoside acetyltransferase.

The applied reaction conditions included an initial 5-min denaturation at 94°C, followed by 30 cycles consisting of denaturation at 94°C for 45 sec, the application of the annealing temperature of each primer for 45 sec, and extension at 72°C for 1 min. After the last reaction, a final extension step of 72°C for 15 min was applied.

To evaluate the reproducibility of the experiments, the PCR reactions were repeated twice for some strains. The strains Klebsiella pneumonia KP15 and Escherichia coli 16-00 were used as positive controls for the genes qnr. The strain E. coli 78-01 and Salmonella Typhimurium 138.12 were used as positive control for the genes aac(6′)-Ib-cr and oqxA/B, respectively. Reactions without DNA as a template were used as negative controls. The PCR products were analyzed through agarose gel electrophoresis and visualized by ultraviolet light after staining the gel with ethidium bromide (1.0 μg/ml).

Determination and analysis of the MIC of ciprofloxacin

MIC of the ciprofloxacin for the 84 Nal^R and 20 Nal^S S. Enteritidis strains studied were determined using Etest^® and the interpretation following the guidelines of the Clinical and Laboratory Standards Institute.⁴³ Strains with MIC ≤0.06 μg/ml were considered sensitive and ≥1 μg/ml resistant.

Results

Detection of the mutations in the QRDR of gyrA

The resulting melting curves for the 84 Nal^R strains and the 20 Nal^S strains allocated the strains into four different melting profiles (Fig. 1). All 20 (100%) of the Nal^S strains were grouped in the same profile named A, and the Nal^R strains comprised the other three profiles. The resistant profile B comprised only one strain, whereas the profile C comprised 15 (17.8%) strains. Profiles D and E were initially grouped in the same melting curve because they presented mutations of complementary bases (A-T and T-A). Thus, a reaction involving the formation of heteroduplexes was necessary to divide these profiles (Fig. 2). After the reaction, the profile D presented 13 (15.4%) strains, and profile E presented 55 (65.4%) strains. The profiles found did not reflect either the source of the strains or the year of isolation.

FIG. 1.

Melting profiles of the 84 nalidixic acid-resistant and 20 nalidixic acid-sensitive Salmonella Enteritidis strains. Black curve: profile A sensitive strains; dashed black curve: profile B resistant strains; gray curve: profile C resistant strains; and dashed gray curve: profile D and E resistant strains.

FIG. 2.

Melting profiles of the D and E profiles after separation through the production of heteroduplexes. Gray curve: profile D resistant strains; and dashed gray curve: profile E resistant strains.

Representative strains of each group were sequenced and exhibited point mutations in the QRDRs of the gene gyrA in Nal^R strains relative to the Nal^S strains. No points of mutation were observed in the genes gyrB, parC, and parE. The points of mutation in the gene gyrA were observed in the codons serine 83 (Ser83), asparagine 87 (Asp87), or serine97 (Ser97) of the sensitive strains.

Profile B presented a single T to C base point mutation in the trio of bases that codes amino acid serine 97 that resulted in a change to proline. Profile C presented a single C to T point mutation in the trio of bases that codes amino acid serine 83 and resulted in a change to phenylalanine. Profile D presented a single G to A point mutation in the trio of bases that codes the aspartic acid 87 and resulted in a change to asparagine. Finally, profile E presented a single G to T point mutation in the trio of bases that codes the amino acid aspartic acid 87 that resulted in a change to tyrosine.

Two (2.5%) Nal^R strains that carried the qnrB gene were both in profile E and did not exhibit differences in susceptibility compared with other strains of the group.

Detection of PMQR genes

None of the 84 Nal^R and the 20 Nal^S strains carried the qnrA, qnrC, qnrD, qnrS, qepA, oqxA, oqxB, or aac(6′)-Ib-cr genes. Only two strains (2.5%) carried the qnrB gene. These strains (107/09 and 191/10) were isolated from chickens in 2009 and 2010.

Determination and analysis of the MIC of ciprofloxacin

A comparison of the MICs of the 84 Nal^R strains according to the different point mutations and the MICs of the 20 Nal^S strains is presented in Table 3. All the 20 (100%) Nal^S strains and 58 (69.0%) Nal^R strains studied were sensitive to ciprofloxacin (MIC ≤0.06 μg/ml), whereas 26 (30.9%) Nal^R strains presented as intermediate to this drug (MIC 0.12 − 0.5 μg/ml). Among the HRMA-resistant profiles, the C (Ser83→Phe) profile exhibited the greatest reduction in susceptibility.

Table 3.

Correlations of the Ciprofloxacin Minimum Inhibitory Concentrations with the gyrA Mutations of the Studied Nalidixic Acid-Sensitive and Resistant Salmonella Enteritidis Strains

	Ciprofloxacin
Strains	MICs average (μg/ml)	Minimum MIC found (μg/ml)	Maximum MIC found (μg/ml)	gyrA mutations	HRMA profile	Mutation's MICs average (μg/ml)
Nalidixic acid sensitive	0.006	0.002	0.008	—	A	0.006
Nalidixic acid resistant	0.09	0.047	0.19	Ser97→Pro	B	0.064
				Ser83→Phe	C	0.138
				Asp87→Tyr	D	0.086
				Asp87→Asn	E	0.091

Asn, aspartic acid; Asp, asparagine; Phe, phenylalanine; Pro, proline; Ser, serine; Tyr, tyrosine.

Discussion

Resistance to quinolones has been increasing in Salmonella in recent years due to the extensive use of fluoroquinolones in both human and veterinary medicine.^7,12 This fact is concerning because treatment failure might occur once nalidixic acid-resistant strains exhibit reduced susceptibility to fluoroquinolones.^10,18,20

In the published studies from our research group based on Salmonella Enteritidis strains isolated in Brazil between 1986 and 2010, nalidixic acid resistance was observed only in the strains isolated after 1996.^29,30 However, a recent analysis with additional strains isolated from 1968 to 1993 and 2012 to 2013 revealed that a single strain isolated in 1969 from humans was resistant to nalidixic acid in addition to three strains isolated in 2012 and 2013 (unpublished data). Thus, our study comprises NAL^R strains isolated over a 44-year period in Brazil and includes strains isolated close to the introduction of nalidixic acid into clinical use in 1962.⁸

The MICs of the fluoroquinolone, ciprofloxacin, for the 84 Salmonella Enteritidis Nal^R strains and the 20 Nal^S strains were determined. The MICs of the strains for ciprofloxacin were all <1 μg/ml, however, even when these MICs were compared in Nal^S and Nal^R strains, the average was 0.006 μg/ml with a range of 0.002 – 0.008 μg/ml for Nal^S strains, whereas for Nal^R strains was 0.09 μg/ml with a range of 0.047 – 0.19 μg/ml (Table 3). These results indicate a reduction in the susceptibility of the Nal^R strains to ciprofloxacin compared with the Nal^S strains. Moreover, among the 84 Nal^R strains, 26 presented an intermediated MIC for ciprofloxacin.

Similar results have been found in studies performed in other countries, such as Spain, Korea, and the United Kingdom, involving Salmonella strains belonging to various serovars, including Enteritidis.^10,44–47 In Brazil, Souza et al. examined strains isolated only in Parana State from 1999 to 2006 and also found reduced susceptibility to ciprofloxacin in the Nal^R strains compared with the Nal^S strains.⁴⁸

The main mechanism of quinolone resistance is thought to be due to target alterations of DNA gyrase and topoisomerase IV. However, a single point mutation in the QRDR of the gyrA usually results in resistance to nalidixic acid. Additional point mutations in gyrA, gyrB, parC, or parE genes typically result in high levels of resistance to these drugs.²⁰

In the present study, only gyrA gene presented points of mutation in the QRDR, which may explain the fact that those strains presented only reduced susceptibility to ciprofloxacin and not resistance. Four different gyrA point mutations were found in the gyrA gene among the Nal^R S. Enteritidis strains relative to the Nal^S strains, and these point mutations resulted in amino acid substitutions in the QRDR of this gene. The most frequent amino acid substitution was Asp87→Tyr, which was present in 55 of the 84 strains studied. Similar results were reported by Escribano et al.⁴⁵ who found this mutation in 18 of 20 Nal^R S. Enteritidis strains isolated in Spain. In contrast, Ferrari et al. studied Salmonella strains isolated in Parana State, Brazil and found that the most common point mutation in the gyrA gene was Asp87→Asn, which was present in 20 of the 64 S. Enteritidis strains studied.³¹ The same frequency was found by Tamang et al., in a study of Salmonella strains isolated in Korea; these authors found the Asp87→Asn mutation in 17 of the 53 strains studied.¹⁰ In the present study, the Asp87→Asn mutation was the third-most common mutation among the Nal^R strains (13 of 84 strains).

Regarding the mutation that produced the greatest decrease in ciprofloxacin susceptibility in the present study, the Ser83→Phe mutation was in 15 of the 84 strains, and the strains with this mutation exhibited an average MIC of 0.138 μg/ml. This mutation was also the same mutation that provided the greatest decrease in ciprofloxacin susceptibility among the strains studied by Tamang et al. that found this mutation only in human strains, which presented an average MIC of 0.25 μg/ml.¹⁰ Furthermore, the other point mutations found in the work mentioned above, that is, Asp87→Asn, Asp87→Gly, and Asp87→Tyr, resulted in similar MICs of ciprofloxacin (0.12–0.25 μg/ml).¹⁰

Other genes, such as those that code efflux pumps, aminoglycoside acetyltransferase, and the plasmid genes qnr, can provide quinolone resistance independently of mutations in the target genes.^19,22,27,28 In the present study, the only PMQR gene found was qnrB, which was present in two of the 84 studied strains. However, these strains also carried the Asp87→Tyr mutation in the gyrA gene, and their MICs did not differ from those of the other strains in the same group that did not carry qnrB, which might suggest that the mechanism that caused quinolone resistance in these cases may be due to the target alteration of the gyrA gene and not qnrB. However, further conjugation experiments should be performed to show that the transfer of plasmid harboring qnrB gene does not change the MIC of the recipient strain.

Similarly, Tamang et al. failed to detect the abovementioned qnr genes in S. Enteritidis strains isolated in Korea.¹⁰ However, other researchers have identified PMQR genes that cause resistance in Salmonella Enteritidis strains. Ferrari et al. detected the qnrA1 gene in one strain of S. Enteritidis that was isolated in Brazil and was resistant to NAL without mutations in the QRDR.³¹ This report was the first of this gene in Salmonella strains from Brazil.³¹ Wasyl et al. found eight S. Enteritidis strains isolated in Poland bearing the genes qnrB or qnrS.⁴⁷ Zhang et al. found 15 S. Enteritidis strains isolated in China that carried the aac(6′)-Ib-cr gene.²⁷

In conclusion, the reduced susceptibility to ciprofloxacin observed in Nal^R strains may cause treatment failures once this drug is commonly used to treat Salmonella infections. Moreover, this reduced susceptibility in these Brazilian strains was provided by target alteration of gene gyrA and not by mobile elements, such as resistance plasmids.

Footnotes

Acknowledgments

The authors thank São Paulo Research Foundation - FAPESP (Proc. 2008/57478-1) for financial support. During the course of this work, F.C. was supported by a scholarship from FAPESP (proc. 2009/09998-9). The authors thank J.D.D. Pitout (University of Calgary, Calgary, Canada) for kindly providing the control strains Kp15 and E. coli 16-00 and Dr. Monique Ribeiro Tiba Casas (Adolfo Lutz Institute, São Paulo, Brazil) for kindly providing the control strain Salmonella Typhimurium 138.12.

Disclosure Statement

No competing financial interests exist.

References

Gantois

, Ducatelle

R.R.

, Pasmans

, Haesebrouck

, Gast

F.R.

, Humphrey

T.J.

, and Van Immerseel

. 2009. Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiol. Rev., 33:718–738.

Humphrey

2004. Salmonella, stress responses and food safety. Nat. Rev. Microbiol., 2:504–509.

Hendriksen

R.S.

, Vieira

A.R.

, Karlsmose

, Lo Fo Wong

D.M.

, Jensen

A.B.

, Wegener

H.C.

, and Aarestrup

F.M.

. 2011. Global monitoring of Salmonella serovar distribution from the World Health Organization Global Foodborne Infections Network Country Data Bank: results of quality assured laboratories from 2001 to 2007. Foodborne Pathog. Dis., 8:887–900.

CDC. 2014. Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2014 (Final Report). CDC, Atlanta, GA.

EFSA. 2015. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-Borne Outbreaks in 2013. EFSA, Parma, Italy, and ECDC, Stockholm, Sweden, p. 165.

Aslam

, Checkley

, Avery

, Chalmers

, Bohaychuk

, Gensler

, Reid-Smith

, and Boerlin

. 2012. Phenotypic and genetic characterization of antimicrobial resistance in Salmonella serovars isolated from retail meats in Alberta, Canada. Food Microbiol., 32:110–117.

Năşcuţiu

A.M.

2012. The tip of the iceberg: quinolone-resistance conferred by mutations in gyrA gene in non-typhoidal Salmonella strains. Roum. Arch. Microbiol. Immunol., 71:17–23.

Wolfson

J.S.

, and Hooper

D.C.

. 1989. Fluoroquinolone antimicrobial agents. Clin. Microbiol. Rev., 2:378–424.

Stevenson

J.E.

, Gay

, Barrett

T.J.

, Medalla

, Chiller

T.M.

, and Angulo

F.J.

. 2007. Increase in nalidixic acid resistance among non-Typhi Salmonella enterica isolates in the United States from 1996 to 2003. Antimicrob. Agents Chemother., 51:195–197.

10.

Tamang

M.D.

, Nam

H.M.

, Kim

, Lee

H.S.

, Kim

T.S.

, Kim

M.J.

, Jang

G.C.

, Jung

S.C.

, and Lim

S.K.

. 2011. Prevalence and mechanisms of quinolone resistance among selected nontyphoid Salmonella isolated from food animals and humans in Korea. Foodborne Pathog. Dis., 8:1199–1206.

11.

Guan

, Xue

, Liu

, Wang

, Jiang

, Zhang

, Yang

, Wang

, et al.. 2013. Plasmid-mediated quinolone resistance—current knowledge and future perspectives. J. Int. Med. Res., 41:20–30.

12.

Phillips

, Casewell

, Cox

, De Groot

, Friis

, Jones

, Nightingale

, Preston

, and Waddell

. 2004. Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data. J. Antimicrob. Chemother., 53:28–52.

13.

Mølbak

, Baggesen

D.L.

, Aarestrup

F.M.

, Ebbesen

J.M.

, Engberg

, Frydendahl

, Gerner-Smidt

, Petersen

A.M.

, and Wegener

H.C.

. 1999. An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica serotype typhimurium DT104. N. Engl. J. Med., 341:1420–1425.

14.

Angulo

F.J.

, Johnson

K.R.

, Tauxe

R.V.

, and Cohen

M.L.

. 2000. Origins and consequences of antimicrobial-resistant nontyphoidal Salmonella: implications for the use of fluoroquinolones in food animals. Microb. Drug. Resist., 6:77–83.

15.

Helms

, Vastrup

, Gerner-Smidt

, and Mølbak

. 2002. Excess mortality associated with antimicrobial drug-resistant Salmonella typhimurium. Emerg. Infect. Dis., 8:490–495.

16.

Choi

S.H.

, Woo

J.H.

, Lee

J.E.

, Park

S.J.

, Choo

E.J.

, Kwak

Y.G.

, Kim

M.N.

, Choi

M.S.

, Lee

N.Y.

, Lee

B.K.

, et al.. 2005. Increasing incidence of quinolone resistance in human non-typhoid Salmonella enterica isolates in Korea and mechanisms involved in quinolone resistance. J. Antimicrob. Chemother., 56:1111–1114.

17.

Bertrand

, Weill

F.X.

, Cloeckaert

, Vrints

, Mairiaux

, Praud

, Dierick

, Wildemauve

, Godard

, Butaye

, et al.. 2006. Clonal emergence of extended-spectrum beta-lactamase (CTX-M-2)-producing Salmonella enterica serovar Virchow isolates with reduced susceptibilities to ciprofloxacin among poultry and humans in Belgium and France (2000 to 2003). J. Clin. Microbiol., 44:2897–2903.

18.

Lee

H.Y.

, Su

L.H.

, Tsai

M.H.

, Kim

S.W.

, Chang

H.H.

, Jung

S.I.

, Park

K.H.

, Perera

, Carlos

, Tan

B.H.

, et al.. 2009. High rate of reduced susceptibility to ciprofloxacin and ceftriaxone among nontyphoid Salmonella clinical isolates in Asia. Antimicrob. Agents Chemother., 53:2696–2699.

19.

Jeong

H.S.

, Kim

J.A.

, Shin

J.H.

, Chang

C.L.

, Jeong

, Cho

J.H.

, Kim

M.N.

, Kim

Y.R.

, Lee

C.H.

, et al.. 2011. Prevalence of plasmid-mediated quinolone resistance and mutations in the gyrase and topoisomerase IV genes in Salmonella isolated from 12 tertiary-care hospitals in Korea. Microb. Drug Resist., 17:551–557.

20.

Crump

J.A.

, Sjölund-Karlsson

, Gordon

M.A.

, and Parry

C.M.

. 2015. Epidemiology, clinical presentation, laboratory diagnosis, antimicrobial resistance, and antimicrobial management of invasive Salmonella infections. Clin. Microbiol. Rev., 28:901–937.

21.

Altekruse

S.F.

, Cohen

M.L.

, and Swerdlow

D.L.

. 1997. Emerging foodborne diseases. Emerg. Infect. Dis., 3:285–293.

22.

Strahilevitz

, Jacoby

G.A.

, Hooper

D.C.

, and Robicsek

. 2009. Plasmid-mediated quinolone resistance: a multifaceted threat. Clin. Microbiol. Rev., 22:664–689.

23.

Kilmartin

, Morris

, O'Hare

, Corbett-Feeney

, and Cormican

. 2005. Clonal expansion may account for high levels of quinolone resistance in Salmonella enterica serovar enteritidis. Appl. Environ. Microbiol., 71:2587–2591.

24.

Hamidian

, Tajbakhsh

, Tohidpour

, Rahbar

, Zali

M.R.

, and Walther-Rasmussen

. 2011. Detection of novel gyrA mutations in nalidixic acid-resistant isolates of Salmonella enterica from patients with diarrhoea. Int. J. Antimicrob. Agents, 37:360–364.

25.

Govinden

, Mocktar

, Moodley

, Sturm

, and Essack

. 2009. Detection of mutations in the gyrA of clinical Salmonella spp. Afr. J. Biotechnol., 8:3911–3914.

26.

, Liao

, Yang

, Sun

, Liu

, Yang

, Ma

, Li

, Zhang

, and Liu

. 2013. Spread of oqxAB in Salmonella enterica serotype Typhimurium predominantly by IncHI2 plasmids. J. Antimicrob. Chemother., 68:2263–2268.

27.

Zhang

, Meng

, Wang

, Xia

, Wang

, Xi

, Meng

, Shi

, Wang

, and Yang

. 2014. Presence of qnr, aac(6′)-Ib, qepA, oqxAB, and mutations in gyrase and topoisomerase in nalidixic acid-resistant Salmonella isolates recovered from retail chicken carcasses. Foodborne Pathog. Dis., 11:698–705.

28.

Kim

E.S.

, Chen

, Braun

, Kim

H.Y.

, Okumura

, Wang

, Jacoby

G.A.

, and Hooper

D.C.

. 2015. Interactions between QnrB, QnrB mutants, and DNA gyrase. Antimicrob. Agents Chemother., 59:5413–5419.

29.

Campioni

, Moratto Bergamini

A.M.

, and Falcão

J.P.

. 2012. Genetic diversity, virulence genes and antimicrobial resistance of Salmonella Enteritidis isolated from food and humans over a 24-year period in Brazil. Food Microbiol., 32:254–264.

30.

Campioni

, Zoldan

M.M.

, and Falcão

J.P.

. 2014. Characterization of Salmonella Enteritidis strains isolated from poultry and farm environments in Brazil. Epidemiol. Infect., 142:1403–1410.

31.

Ferrari

, Galiana

, Cremades

, Rodríguez

J.C.

, Magnani

, Tognim

M.C.

, Oliveira

T.C.

, and Royo

. 2013. Plasmid-mediated quinolone resistance (PMQR) and mutations in the topoisomerase genes of Salmonella enterica strains from Brazil. Braz. J. Microbiol., 44:651–656.

32.

Souza

R.A.

, and Falcão

J.P.

. 2012. A novel high-resolution melting analysis-based method for Yersinia pseudotuberculosis genotyping. J. Microbiol. Methods, 91:329–335.

33.

Slinger

, Bellfoy

, Desjardins

, and Chan

. 2007. High-resolution melting assay for the detection of gyrA mutations causing quinolone resistance in Salmonella enterica serovars Typhi and Paratyphi. Diagn. Microbiol. Infect. Dis., 57:455–458.

34.

Smith

B.L.

, Lu

C.P.

, and Alvarado Bremer

J.R.

. 2010. High-resolution melting analysis (HRMA): a highly sensitive inexpensive genotyping alternative for population studies. Mol. Ecol. Resour., 10:193–196.

35.

Souza

R.A.

, and Falcão

J.P.

. 2014. A novel high-resolution melting analysis-based method for Yersinia enterocolitica genotyping. J. Microbiol. Methods, 106:129–134.

36.

Souza

R.A.

, Pitondo-Silva

, Falcão

D.P.

, and Falcão

J.P.

. 2010. Evaluation of four molecular typing methodologies as tools for determining taxonomy relations and for identifying species among Yersinia isolates. J. Microbiol. Methods, 82:141–150.

37.

Fàbrega

, du Merle

, Le Bouguénec

, Jiménez de Anta

M.T.

, and Vila

. 2009. Repression of invasion genes and decreased invasion in a high-level fluoroquinolone-resistant Salmonella typhimurium mutant. PLoS One, 4:e8029.

38.

Campioni

, and Falcão

J.P.

. 2014. Genotypic diversity and virulence markers of Yersinia enterocolitica biotype 1A strains isolated from clinical and non-clinical origins. APMIS, 122:215–222.

39.

Park

C.H.

, Robicsek

, Jacoby

G.A.

, Sahm

, and Hooper

D.C.

. 2006. Prevalence in the United States of aac(6′)-Ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob. Agents Chemother., 50:3953–3955.

40.

Jacoby

G.A.

, Gacharna

, Black

T.A.

, Miller

G.H.

, and Hooper

D.C.

. 2009. Temporal appearance of plasmid-mediated quinolone resistance genes. Antimicrob. Agents Chemother., 53:1665–1666.

41.

Kim

H.B.

, Park

C.H.

, Kim

C.J.

, Kim

E.C.

, Jacoby

G.A.

, and Hooper

D.C.

. 2009. Prevalence of plasmid-mediated quinolone resistance determinants over a 9-year period. Antimicrob. Agents Chemother., 53:639–645.

42.

Chen

, Zhang

, Pan

, Yin

, Pan

, Gao

, and Jiao

. 2012. Prevalence of qnr, aac(6′)-Ib-cr, qepA, and oqxAB in Escherichia coli isolates from humans, animals, and the environment. Antimicrob. Agents Chemother., 56:3423–3427.

43.

CLSI. 2016. Performance Standards for Antimicrobial Susceptibility Testing. CLSI Supplement M100S. Clinical and Laboratory Standards Institute, Wayne, PA.

44.

Griggs

D.J.

, Gensberg

, and Piddock

L.J.

. 1996. Mutations in gyrA gene of quinolone-resistant Salmonella serotypes isolated from humans and animals. Antimicrob. Agents Chemother., 40:1009–1013.

45.

Escribano

, Rodríguez

J.C.

, and Royo

. 2004. Mutations in the gyrA gene in Salmonella enterica clinical isolates with decreased ciprofloxacin susceptibility. Int. J. Antimicrob. Agents, 24:300–303.

46.

San Martín

, Lapierre

, Toro

, Bravo

, Cornejo

, Hormazabal

J.C.

, and Borie

. 2005. Isolation and molecular characterization of quinolone resistant Salmonella spp. from poultry farms. Vet. Microbiol., 110:239–244.

47.

Wasyl

, Hoszowski

, and Zając

. 2014. Prevalence and characterisation of quinolone resistance mechanisms in Salmonella spp. Vet. Microbiol., 171:307–314.

48.

Souza

R.B.

, Ferrari

R.G.

, Magnani

, Kottwitz

L.B.

, Alcocer

, Tognim

M.C.

, and Oliveira

T.C.

. 2010. Ciprofloxacin susceptibility reduction of Salmonella strains isolated from outbreaks. Braz. J. Microbiol., 41:497–500.