Open Access

Epidemic characterization and molecular genotyping of Shigella flexneri isolated from calves with diarrhea in Northwest China

  • Zhen Zhu1,
  • Mingze Cao1,
  • Xuzheng Zhou1,
  • Bing Li1 and
  • Jiyu Zhang1Email author
Contributed equally
Antimicrobial Resistance & Infection Control20176:92

https://doi.org/10.1186/s13756-017-0252-6

Received: 27 June 2017

Accepted: 30 August 2017

Published: 6 September 2017

Abstract

Background

The widespread presence of antibiotics resistance genes in pathogenic bacteria can cause enormous problems. Food animals are one of the main reservoirs of intestinal pathogens that pose a potential risk to human. Analyzing the epidemiological characteristics and resistance patterns of Shigella flexneri in calves is necessary for animal and human health.

Methods and results

A total of 54 Shigella flexneri isolates, including six serotypes (1a, 2a, 2b, 4a, 6 and Xv), were collected from 837 fecal samples obtained from 2014 to 2016. We performed pulsed-field gel electrophoresis (PFGE) and applied the restriction enzyme NotI to analyze the genetic relatedness among the 54 isolates and to categorize them into 31 reproducible and unique PFGE patterns. According to the results of antimicrobial susceptibility tests, all 26 Shigella flexneri 2a serotypes were resistant to cephalosporin and/or fluoroquinolones. The genes bla TEM-1 , bla OXA-1 , and bla CTX-M-14 were detected in 19 cephalosporin-resistant S. flexneri 2a isolates. Among 14 fluoroquinolone-resistant isolates, the aac(6)-Ib-cr gene was largely present in each strain, followed by qnrS (5). Only one ciprofloxacin-resistant isolate harbored the qepA gene. Sequencing the quinolone resistance determining regions (QRDRs) of the fluoroquinolone-resistant isolates revealed two point mutations in gyrA (S83 L, D87N/Y) and a single point mutation in parC (S80I). Interestingly, two gyrA (D87N/Y) strains were resistant to ciprofloxacin.

Conclusions

The current study enhances our knowledge of Shigella in cattle, although continual surveillance is necessary for the control of shigellosis. The high level of cephalosporin and/or fluoroquinolone resistance in Shigella warns us of a potential risk to human and animal health.

Keywords

Shigella flexneri Antimicrobial susceptibility Resistant

Background

The majority of Enterobactericeae family bacteria, including Salmonella, E. coli and Shigella spp., the major etiological agent of diarrheal disease, are a global public health burden, particularly in low-income countries [13]. Shigella is phylogenetically distinct from several independent E. coli strains and has evolved through convergent evolution [4]. The genus Shigella consists of four subgroups differentiated according to their biochemical and serological properties: A (S. dysenteriae), B (S. flexneri), C (S. boydii), and D (S. sonnei). All four species of Shigella cause shigellosis, but S. flexneri is the predominant subgroup found in developing countries, whereas S. sonnei is found in industrialized countries [5]. The first Shigella species identified was S. dysenteriae, followed by S. flexneri at the end of the 19th century. Shigellosis became a notorious and widespread epidemic during World War 1 with the transmission of S. flexneri strain NCTC1, a 2a lineage [6, 7]. Based on the differing structural characteristics of the antigenic determinants of the O antigen, S. flexneri is divided into no fewer than 20 serotypes: 1a, 1b, 1c, 1d, 2a, 2b, 2v, 3a, 3b, 4a, 4av, 4b, 4c, 5a, 5b, X, Xv, Y, Yv, 6, and 7b [8, 9].

Given that shigellosis is a global public health burden, previous studies have focused on the human gastrointestinal pathogens but have ignored animal groups. Shigella spp. infect and also cause corresponding clinical symptoms in monkeys, cows, pigs, chickens and other animals [1013]. Indeed, animals that live in environments characterized by poor sanitary hygiene, restricted access to clean drinking water and long-term exposure to contaminated food are prone to dysentery [14, 15]. Many antibiotics are used to control disease and promote growth during the breeding process, leading to the widespread dissemination of antibiotic resistance genes (ARGs). The spread of drug resistance among pathogenic bacteria in humans and animals may be disastrous.

The present study investigated the Shigella epidemic in cows in the northwest region of China. S. flexneri 2a was first isolated from a yak with diarrhea in Tibet in 2014. In this study, we used pulsed-field gel electrophoresis (PFGE) to analyze the relationships among S. flexneri isolates and tested for antimicrobial susceptibility patterns. Our results will help prevent diarrhea in calves and will assist in the selection of effective antibiotics against Shigella.

Methods

Bacterial isolation and identification

Fresh stool samples were isolated from 2014 to 2016 in Northwest China (Gansun, Shanxi, Qinghai, Xinjiang and Tibet) from calves (3 to 20 days) with diarrhea. Samples were stored in transport medium, cultured directly on Salmonella-Shigella (SS) agar and incubated at 37 °C for 24 h to select for Shigella. Resultant colonies (colorless, semitransparent, smooth, and moist circular) [16] were picked and grown at 37 °C for 24 h on MacConkey (MAC) Agar to verify identity. Colonies were selected and cultured in brain heart infusion broth at 37 °C for 5 h with shaking at 250 rpm. All isolates were confirmed using API20E test strips (bioMerieux Vitek, Marcy-l’ Etoile, France) according to the manufacturer’s recommendations. Shigella was serotyped using a commercially available kit (Denka Seiken, Tokyo, Japan) and confirmed by PCR [17].

Antimicrobial susceptibility testing

The antimicrobial susceptibility of S. flexneri isolates was determined via the Kirby–Bauer disc-diffusion method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [18].

The antibiotic discs (OXOID, UK) included penicillin G (P, 10 μg), ampicillin (AMP, 10 μg), amoxicillin/clavulanic acid (AMC, 30 μg), cephalothin (KF, 30 μg), cephazolin (KZ, 30 μg), cefamandole (MA, 30 μg), cefoxitin (FOX, 30 μg), ceftriaxone (CRO, 30 μg), cefotaxime (CTX, 30 μg), cefoperazone (CFP, 75 μg), cefepime (FEP, 30 μg), meropenem (MEM, 10 μg), imipenem (IPM, 10 μg), norfloxacin (NOR, 10 μg), enrofloxacin (ENR, 5 μg), levofloxacin (LEV, 5 μg), ciprofloxacin (CIP, 5 μg), erythromycin (E, 15 μg), chloramphenicol (C, 30 μg), tetracycline (TE, 30 μg), streptomycin (S, 10 μg), gentamicin (CN, 10 μg), and amikacin (AK, 30 μg). E. coli strain ATCC25922 was used as a quality control strain in each test batch.

PCR amplification of ARGs

We performed PCR assays that targeted 24 different ARGs using the primers described in Table 1. To determine the underlying resistance mechanism of β-lactam antibiotics, we amplified extended-spectrum β-lactamase (ESBL) genes, specifically bla CTX-M , bla SHV , bla TEM , and bla OXA , as well as ampC genes, specifically bla MOX , bla FOX , bla DHA , bla CIT , bla ACC , and bla MIR [1921]. Plasmid-mediated quinolone resistance (PMQR) determinant genes, including qnrA, qnrB, qnrD, qnrS, qepA and aac(6′)-Ib-cr and four quinolone resistance determining region (QRDR) genes as well as DNA gyrase (gyrA,gyrB) and topoisomerase IV (parC,parE) were amplified to determine the underlying mechanism of quinolone resistance [16, 2123]. The PCR fragments were sequenced after purification and compared to sequences in GenBank.
Table 1

Primers for the detection of antibiotic resistance genes

Target

Primer sequence (5′ to 3′)

Amplicon size (bp)

Reference

β-lactamase

bla CTX-M-1

F: GGTTAAAAAATCACTGCGTC

873

Cui et al., 2015 [16]

R: TTACAAACCGTCGGTGACGA

bla CTX-M-2

F: CGACGCTACCCCTGCTATT

552

Zong et al., 2008 [17]

R: CCAGCGTCAGATTTTTCAGG

bla CTX-M-8

F: TCGCGTTAAGCGGATGATGC

689

Zong et al., 2008 [17]

R: AACCCACGATGTGGGTAGC

bla CTX-M-9

F: AGAGTGCAACGGATGATG

868

Cui et al., 2015 [16]

R: CCAGTTACAGCCCTTCGG

bla CTX-M-25

F: TTGTTGAGTCAGCGGGTTGA

497

Liu et al., 2015 [18]

R: GCGCGACCTTCCGGCCAAAT

bla SHV

F: CGCCGGGTTATTCTTATTTGTCGC

1015

Zong et al., 2008 [17]

R: TCTTTCCGATGCCGCCGCCAGTCA

bla TEM

F: ATGAGTATTCAACTTTCCG

876

This study

R: CCAATGCTTAATCAGTGAG

bla OXA

F: ATTAAGCCCTTTACCAAACCA

890

Cui et al., 2015 [16]

R: AAGGGTTGGGCGATTTTGCCA

bla MOX

F: GCTGCTCAAGGAGCACAGGAT

520

Cui et al., 2015 [16]

R: CACATTGACATAGGTGTGGTGC

bla FOX

F: AACATGGGGTATCAGGGAGATG

190

Cui et al., 2015 [16]

R: CAAAGCGCGTAACCGGATTGG

bla DHA

F: AACTTTCACAGGTGTGCTGGGT

405

Cui et al., 2015 [16]

R: CCGTACGCATACTGGCTTTGC

bla CIT

F: TGGCCAGAACTGACAGGCAAA

462

Cui et al., 2015 [16]

R: TTTCTCCTGAACGTGGCTGGC

bla ACC

F: AACAGCCTCAGCAGCCGGTTA

346

Cui et al., 2015 [16]

R: TTCGCCGCAATCATCCCTAGC

bla MIR

F: TCGGTAAAGCCGATGTTGCGG

302

Cui et al., 2015 [16]

R: CTTCCACTGCGGCTGCCAGTT

PMQRs

qnrA

F: ATTTCTCACGCCAGGATTTG

516

Colobatiu et al.,2015 [19]

R: GATCGGCAAAGGTTAGGTCA

qnrB

F: GATCGTGAAAGCCAGAAAGG

476

Colobatiu et al.,2015 [19]

R: ACGATGCCTGGTAGTTGTCC

qnrD

F: CGAGATCAATTTACGGGGAATA

656

Cui et al.,2015 [13]

R: AACAAGCTGAAGCGCCTG

qnrS

F: ACGACATTCGTCAACTGCAA

417

Colobatiu et al.,2015 [19]

R: TAAATTGGCACCCTGTAGGC

aac(6′)-Ib-cr

F: CCCGCTTTCTCGTAGCA

544

Colobatiu et al.,2015 [19]

R: TTAGGCATCACTGCGTCTTC

qepA

F: CGTGTTGCTGGAGTTCTTC

403

Colobatiu et al.,2015 [19]

R: CTGCAGGTACTGCGTCATG

QRDR

gyrA

F: TACACCGGTCAACATTGAGG

648

Hu et al.,2007 [20]

R: TTAATGATTGCCGCCGTCGG

gyrB

F: TGAAATGACCCGCCGTAAAGG

309

Hu et al.,2007 [20]

R: GCTGTGATAACGCAGTTTGTCCGGG

parC

F: GTACGTGATCATGGACCGTG

531

Hu et al.,2007 [20]

R: TTCGGCTGGTCGATTAATGC

parE

F: ATGCGTGCGGCTAAAAAAGTG

290

Hu et al.,2007 [20]

R: TCGTCGCTGTCAGGATCGATAC

PFGE

Genotypes and transmission patterns were determined by performing PFGE according to the method described in a previous study [19]. S. flexneri isolates were digested with the restriction enzyme NotI (TaKaRa, Japan) at 37 °C for 3 h to generate a DNA fingerprinting profile. Salmonella enterica serotype Braenderup strain H9812 was digested with XbaI (TaKaRa, Japan) and used as a molecular size standard. Electrophoresis was performed on the CHEF Mapper XA system (Bio-Rad) with a 1% agarose SeaKem Gold gel (Lonza, USA). Electrophoretic parameters were determined by performing multiple screening runs and included switching times of 2.16 to 54.17 s, a voltage of 6 v/cm, a 120° angle and a run time of 21 h. PFGE images were obtained using a Universal Hood II (Bio-RAD, USA) and analyzed using BioNumerics software version 7.1 (Applied Maths, Sint-Martens-Latem, Belgium). A clustering tree that indicated relative genetic similarity was constructed using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and the Dice-predicted similarity value with a 1.0% pattern optimization and 1.5% band position tolerance.

Results

Bacterial isolation and identification

During our epidemiological survey of Shigella, we collected 873 fecal samples from calves with diarrhea and obtained 54 S. flexneri isolates from five provinces in northwest China from 2014 to 2016. Isolate information is shown in detail in Table 2. Among the 54 S. flexneri isolates, there were six serotypes: five (9.26%) isolates were 1a, twenty-six (48.15%) isolates were 2a, four (7.41%) isolates were 2b, six (11.11%) isolates were 4a, eight (14.81%) isolates were 6, and five (9.26%) isolates were Xv (Fig. 1). Our surveillance of the Gansu isolates identified all of the serotypes, except 4a. All 4a serotypes were isolated from Shanxi, while all Xv and 1a serotypes were from Gansu. Additionally, serotype 2a was widely isolated from each province, with the exception of Xinjiang, and serotype 6 was found only in yaks. Interestingly, Shigella was primarily isolated in the first quarter and fourth quarter, accounting for 54% (29/54) and 30% (16/54), respectively (Fig. 2).
Table 2

Strain information of S. flexneri isolates from diarrheal calves, 2014 to 2016

Strain name

Serotype

Isolation year

Origin

Province

TYSF1412001

2a

2014

Yak

Tibet

GBSF1412056

2a

2014

Beef cattle

Gansu

GBSF1501026

2a

2015

Beef cattle

Gansu

GBSF1501071

Xv

2015

Beef cattle

Gansu

GYSF1501076

6

2015

Yak

Gansu

QYSF1501088

6

2015

Yak

Qinghai

XBSF1501093

2b

2015

Beef cattle

Xinjiang

GBSF1501105

2a

2015

Beef cattle

Gansu

SBSF1501123

4a

2015

Beef cattle

Shanxi

QYSF1502130

6

2015

Yak

Qinghai

GBSF1502176

2a

2015

Beef cattle

Gansu

GYSF1502197

6

2015

Yak

Gansu

SBSF1502219

4a

2015

Beef cattle

Shanxi

XBSF1502236

2b

2015

Beef cattle

Xinjiang

GBSF1503241

2a

2015

Beef cattle

Gansu

GYSF1503270

1a

2015

Yak

Gansu

GBSF1503288

1a

2015

Beef cattle

Gansu

GBSF1505314

2a

2015

Beef cattle

Gansu

SBSF1505331

2a

2015

Beef cattle

Shanxi

GBSF1506340

Xv

2015

Beef cattle

Gansu

GBSF1507358

1a

2015

Beef cattle

Gansu

GBSF1509369

2a

2015

Beef cattle

Gansu

GBSF1510375

2a

2015

Beef cattle

Gansu

GBSF1510390

2a

2015

Beef cattle

Gansu

QYSF1511395

2a

2015

Yak

Qinghai

GBSF1511401

2a

2015

Beef cattle

Gansu

GYSF1511409

2a

2015

Yak

Gansu

SBSF1512413

4a

2015

Beef cattle

Shanxi

GBSF1512419

2b

2015

Beef cattle

Gansu

GBSF1512425

2a

2015

Beef cattle

Gansu

GBSF1512433

2a

2015

Beef cattle

Gansu

GBSF1601015

Xv

2016

Beef cattle

Gansu

GBSF1601024

Xv

2016

Beef cattle

Gansu

TYSF1601031

2b

2016

Yak

Tibet

GBSF1601064

2a

2016

Beef cattle

Gansu

GYSF1601073

6

2016

Yak

Gansu

GBSF1602082

2a

2016

Beef cattle

Gansu

QYSF1602094

6

2016

Yak

Qinghai

GBSF1602098

2a

2016

Beef cattle

Gansu

GBSF1602103

2a

2016

Beef cattle

Gansu

SBSF1603115

4a

2016

Beef cattle

Shanxi

SBSF1603121

4a

2016

Beef cattle

Shanxi

GBSF1603138

2a

2016

Beef cattle

Gansu

GBSF1603149

2a

2016

Beef cattle

Gansu

QYSF1603158

6

2016

Yak

Qinghai

SBSF1604173

2a

2016

Beef cattle

Shanxi

SBSF1604195

4a

2016

Beef cattle

Shanxi

GBSF1605203

Xv

2016

Beef cattle

Gansu

GBSF1608241

2a

2016

Beef cattle

Gansu

GYSF1610256

2a

2016

Yak

Gansu

GYSF1610266

6

2016

Yak

Gansu

GBSF1610275

1a

2016

Beef cattle

Gansu

GBSF1611283

1a

2016

Beef cattle

Gansu

GBSF1611290

2a

2016

Beef cattle

Gansu

Fig. 1

S. flexneri serotypes collected from 2014 to 2016

Fig. 2

Number of S. flexneri isolated from different quarters

Antimicrobial susceptibility testing

The 54 S. flexneri isolates were examined for susceptibility to 23 antibiotics. More than 50% of isolates were resistant to 8 antibiotics. Among them, resistance to P (54/54, 100%), AMP (51/54, 94.44%) and TE (49/54, 90.74) was most common, followed by E (46/54, 85.19%), S (38/54, 70.37%), KZ (34/54, 62.96%), KF (29/54, 53.70%) and CN (29/54, 53.70%). None of the isolates were resistant to IMP, MEM and the fourth-generation cephalosporin FEP. In addition, although a certain number of isolates were resistant to second- and third-generation cephalosporins (MA, FOX, CRO, CTX and CFP) and fluoroquinolones (CIP, NOR, ENR and LEV), these comprised no more than 30% of the total number of isolates, and the resistance rate was lower than those of other antibiotics (Table 3, Fig. 3).
Table 3

Statistical analysis of the results of antimicrobial susceptibility to 23 antibiotics for 54 S. flexneri

Antibiotic

Antimicrobial resistance rate No. (%)

Total (n = 54)

Gansu (n = 37)

Shanxi (n = 8)

Xinjiang (n = 2)

Qinghai (n = 5)

Tibet (n = 2)

Penicillin G (P)

54 (100%)

37 (100%)

8 (100%)

2 (100%)

5 (100%)

2 (100%)

Ampicillin (AMP)

51 (94.44%)

37 (100%)

8 (100%)

2 (100%)

3 (60%)

1 (50%)

Amoxycillin/Clavulanic acid (AMC)

5 (9.62%)

3 (8.11%)

1 (12.5%)

1 (50%)

0

0

Cephalothin (KF)

29 (53.70%)

19 (51.35%)

5 (62.5%)

2 (100%)

2 (40%)

1 (50%)

Cephazolin (KZ)

34 (62.96%)

21 (56.76%)

7 (87.5%)

2 (100%)

3 (60%)

1 (50%)

Cefamandole (MA)

16 (29.63%)

12 (32.43%)

2 (25%)

1 (50%)

1 (20%)

0

Cefoxitin (FOX)

3 (5.56%)

2 (5.41%)

1 (12.5%)

0

0

0

Ceftriaxone (CRO)

12 (22.22%)

9 (24.32%)

2 (25%)

1 (50%)

0

0

Cefotaxime (CTX)

14 (25.93%)

10 (27.03%)

2 (25%)

1 (50%)

1 (20%)

0

Cefoperazone (CFP)

6 (11.11%)

6 (16.22%)

0

0

0

0

Cefepime (FEP)

0

0

0

0

0

0

Meropenem (MEM)

0

0

0

0

0

0

Imipenem (IPM)

0

0

0

0

0

0

Norfloxacin (NOR)

16 (29.63%)

12 (32.43%)

3 (37.5%)

1 (50%)

0

0

Enrofloxacin (ENR)

13(24.07%)

11 (29.73%)

2 (25%)

0

0

0

Levofloxacin (LEV)

14 (25.93%)

11 (29.73%)

2 (25%)

1 (50%)

0

0

Ciprofloxacin (CIP)

2 (3.70%)

2 (5.41%)

0

0

0

0

Erythromycin (E)

46 (85.19%)

35 (94.59%)

6 (75%)

2 (100%)

3 (60%)

0

Tetracycline (TE)

49 (90.74%)

35 (94.59%)

8 (100%)

2 (100%)

3 (60%)

1 (50%)

Chloramphenicol (C)

17 (31.48%)

10 (27.03%)

6 (75%)

1 (50%)

0

0

Streptomycin (S)

38 (70.37%)

30 (81.08%)

4 (50%)

2 (100%)

2 (40%)

0

Gentamicin (CN)

29 (53.70%)

23 (62.16%)

4 (50%)

2 (100%)

0

0

Amikacin (AK)

3 (5.56%)

3 (8.11%)

0

0

0

0

Fig. 3

PFGE dendrogram and antibiotic susceptibility profile of 54 NotI-digested S. flexneri. B = Beef cattle; Y = Yak. R = Resistance; N = Sensitive and Intermediary

Remarkably, all 26 S. flexneri 2a isolates demonstrated varying degrees of resistance to cephalosporins and/or fluoroquinolones and exhibited multidrug resistance (MDR). The S. flexneri 2a isolates were resistant to 14 diverse cephalosporins/fluoroquinolones. Among them, 73.06% (19/26) of isolates were resistant to cephalosporin, 53.85% (14/26) of isolates were resistant to fluoroquinolones, and 26.92% (7/26) of isolates were resistant to both cephalosporin and fluoroquinolones. Furthermore, isolate GBSF1512433 was resistant to all cephalosporins (with the exception of FEP) and fluoroquinolones (with the exception of CIP). Compared with the S. flexneri 2a isolates collected from beef calves, the 4 yak isolates were sensitive to most cephalosporins and fluoroquinolones but resistant to KF, KZ and MA (Table 4).
Table 4

Statistical analysis of the cephalosporin and/or fluoroquinolone susceptibility for 26 S. flexneri 2a

Cephalosporin and/or Fluorquinolones resistance spectrum

Cephalosporin and/or Fluorquinolones resistance rate No. (%)

Total (n = 26)

Gansu (n = 22a)

Shanxi (n = 2)

Qinghai (n = 1a)

Tibet (n = 1a)

KF/KZ

5 (19.23%)

3 (13.64%)

0

1a (100%)

1a (100%)

KF/KZ/MA

3 (11.54%)

2a (9.09%)

1 (50%)

0

0

KF/KZ/MA/CRO

1 (3.85%)

1 (4.55%)

0

0

0

KF/KZ/MA/CTX

1 (3.85%)

1 (4.55%)

0

0

0

KF/KZ/MA/CRO/CFP

1 (3.85%)

1 (4.55%)

0

0

0

KF/KZ/MA/FOX/CRO/CTX/CFP

1 (3.85%)

1 (4.55%)

0

0

0

NOR/LEV

3 (11.54%)

3 (13.64%)

0

0

0

ENR/LEV

3 (11.54%)

3 (13.64%)

0

0

0

NOR/ENR/LEV/CIP

1 (3.85%)

1 (4.55%)

0

0

0

KF/KZ/MA/CRO/CTX/NOR/LEV

2 (7.69%)

1 (4.55%)

1 (50%)

0

0

KF/KZ/MA/CRO/CTX/NOR/ENR/LEV

1 (3.85%)

1 (4.55%)

0

0

0

KF/KZ/MA/CTX/CFP/CIP/NOR/ENR

2 (7.69%)

2 (9.09%)

0

0

0

KF/KZ/MA/CRO/CTX/CFP/NOR/ENR/CIP

1 (3.85%)

1 (4.55%)

0

0

0

KF/KZ/MA/FOX/CRO/CTX/CFP/NOR/ENR/LEV

1 (3.85%)

1 (4.55%)

0

0

0

aa yak origin S. flexneri 2a isolate

ARGs analysis of cephalosporin- and/or fluoroquinolone-resistant S. flexneri 2a isolates

In this study, only three β-lactamase gene types (bla OXA-1 , bla TEM-1 and bla CTX-M-14 ) were identified among the 19 cephalosporin-resistant S. flexneri 2a isolates (Table 5). All isolates harbored bla TEM-1 type ARGs (100%), 15 isolates harbored bla OXA-1 (15/19, 78.95%), and 14 harbored bla CTX-M-14 (14/19, 73.68%). In total, 63.16% (12/19) of isolates harbored three β-lactamase gene types. All S. flexneri 2a isolates from yaks were negative for bla CTX-M type ARGs.
Table 5

Antimicrobial spectrum and ARGs analysis of S. flexneri 2a with resistance to cephalosporin

Strain name

Antimicrobial spectrum

ARGs in plasmid

TEM

OXA

CTX-M-9

TYSF1412001

KF/KZ

TEM-1

OXA-1

----

QYSF1511395

KF/KZ

TEM-1

----

----

GBSF1503241

KF/KZ

TEM-1

OXA-1

CTX-M-14

GBSF1502176

KF/KZ

TEM-1

OXA-1

CTX-M-14

GBSF1602082

KF/KZ

TEM-1

----

CTX-M-14

SBSF1505331

KF/KZ/MA

TEM-1

OXA-1

CTX-M-14

GYSF1511409

KF/KZ/MA

TEM-1

----

----

GYSF1610256

KF/KZ/MA

TEM-1

OXA-1

----

GBSF1510375

KF/KZ/MA/CRO

TEM-1

OXA-1

CTX-M-14

GBSF1501105

KF/KZ/MA/CTX

TEM-1

OXA-1

CTX-M-14

GBSF1412056

KF/KZ/MA/CRO/CFP

TEM-1

OXA-1

CTX-M-14

GBSF1602103

KF/KZ/MA/FOX/CRO/CTX/CFP

TEM-1

OXA-1

CTX-M-14

GBSF1601064

KF/KZ/MA/CRO/CTX/NOR/LEV

TEM-1

----

CTX-M-14

SBSF1604173

KF/KZ/MA/CRO/CTX/NOR/LEV

TEM-1

OXA-1

CTX-M-14

GBSF1611290

KF/KZ/MA/CTX/CFP/NOR/ENR

TEM-1

OXA-1

CTX-M-14

GBSF1501026

KF/KZ/MA/CTX/CFP/NOR/ENR

TEM-1

OXA-1

CTX-M-14

GBSF1512425

KF/KZ/MA/CRO/CTX/NOR/ENR/LEV

TEM-1

OXA-1

----

GBSF1602098

KF/KZ/MA/CRO/CTX/CFP/NOR/ENR/CIP

TEM-1

OXA-1

CTX-M-14

GBSF1512433

KF/KZ/MA/FOX/CRO/CTX/CFP/NOR/ENR/LEV

TEM-1

OXA-1

CTX-M-14

Both PMQR genes and SNPs in QRDRs were identified for 14 quinolone-resistant isolates (Table 6). According to the PCR results, all quinolone-resistant isolates were positive for aac(6)-Ib-cr but negative for qepA, except strain GBSF1602098. Only five (5/14, 35.71%) strains isolated from Gansu harbored qnrS, and no isolate harbored all three ARGs simultaneously. The point mutations in the QRDR genes play important roles in determining quinolone and/or fluoroquinolone resistance [24]. In the present study, we successfully amplified all four QRDR genes and compared them to reference sequences. We found two point mutations in gyrA and one point mutation each in gyrA and parC (Table 6). All quinolone-resistant strains carried mutations that altered the amino acid sequences of gyrA (S83 L) and parC (S80I). In addition, each strain carried the mutation 87 (D → N or Y) in gyrA, with the exception of GBSF1510390. Interestingly, GBSF1505314 and GBSF1602098 harbored the gyrA D87Y mutation, which confers resistance to ciprofloxacin.
Table 6

Antimicrobial spectrum and amino acid types in QRDR and PMQRs genes analysis of S. flexneri 2a with resistance to fluoroquinolones

Strain name

Antimicrobial spectrum

QRDR

ARGs in plasmid

gyrA

parC

aac(6)-Ib-cr

qnrS

qepA

83

87

80

GBSF1509369

NOR/LEV

S83 L

D87N

S80I

+

GBSF1511401

NOR/LEV

S83 L

D87N

S80I

+

GBSF1608241

NOR/LEV

S83 L

D87N

S80I

+

GBSF1510390

ENR/LEV

S83 L

D87D

S80I

+

+

GBSF1603138

ENR/LEV

S83 L

D87N

S80I

+

GBSF1603149

ENR/LEV

S83 L

D87N

S80I

+

GBSF1505314

NOR/ENR/LEV/CIP

S83 L

D87Y

S80I

+

+

GBSF1601064

KF/KZ/MA/CRO/CTX/NOR/LEV

S83 L

D87N

S80I

+

SBSF1604173

KF/KZ/MA/CRO/CTX/NOR/LEV

S83 L

D87N

S80I

+

GBSF1611290

KF/KZ/MA/CTX/CFP/NOR/ENR

S83 L

D87N

S80I

+

GBSF1501026

KF/KZ/MA/CTX/CFP/NOR/ENR

S83 L

D87N

S80I

+

+

GBSF1512425

KF/KZ/MA/CRO/CTX/NOR/ENR/LEV

S83 L

D87N

S80I

+

+

GBSF1602098

KF/KZ/MA/CRO/CTX/CFP/NOR/ENR/CIP

S83 L

D87Y

S80I

+

+

GBSF1512433

KF/KZ/MA/FOX/CRO/CTX/CFP/NOR/ENR/LEV

S83 L

D87N

S80I

+

+

+: Presence corresponding genes

-: Absence corresponding genes

PFGE pattern analysis

PFGE was performed to determine the genetic relatedness among the isolates and to study the molecular epidemiology in specific geographical regions [25]. The PFGE patterns of the 54 NotI-digested S. flexneri isolates were heterogeneous, and multiple PFGE patterns were present among the strains. Thus, diverse factors such as geography and environment may affect PFGE patterns. At an 80% similarity level, S. flexneri isolates generated 31 reproducible and unique PFGE patterns, including 11 common types (CT) and 20 single types (ST) (Fig. 3).

Among all isolates, the majority of S. flexneri 2a (26/54, 48.15%) isolates were classified into 11 PFGE patterns (4 CT and 7 ST). These PFGE patterns were closely related to each other, except the Tibet (TYSF1412001) and Qinghai (QYSF1511395) isolates, suggesting the strains isolated from different geographical locations exhibit diverse PFGE patterns and a capricious genetic diversity.

Discussion

ARGs are widespread and cause problems when present in pathogens [26]. Over the past decade, MDR Shigella has been reported in many countries [27]. However, only a few studies have described the prevalence of Shigella in animals worldwide. In the present study, we investigated the epidemiology of S. flexneri in cows in northwest China. During a 2-year survey, 54 S. flexneri isolates were obtained. Unfortunately, 16S rRNA gene sequence analysis does not effectively distinguish between closely related strains in a superfamily, such as Shigella and E. coli [28], and conventional biochemical and serological techniques are also insufficient. Therefore, PFGE was utilized to analyze the molecular characteristics of these isolates, to determine the relatedness among isolates and to study the molecular epidemiology in specific geographical regions. The clustering results allowed us to analyze the epidemiological trends of S. flexneri. Characterization of these isolates will be helpful for clinical diagnosis, treatment, prevention and the control of shigellosis [15].

Antimicrobial resistance has emerged as a serious problem [29], particularly for conventional, older-generation antibiotics such as P, AMP, TE, and E. According to the results of our antimicrobial susceptibility tests, cephalosporin and fluoroquinolone resistance rates in our isolates were higher than those in human isolates [19, 26]. Notably, the predominant S. flexneri 2a isolates were all resistant to cephalosporins, fluoroquinolones and multiple antibiotics. Two isolates (GBSF1505314 and GBSF1602098) were also resistant to ciprofloxacin, which is the first-line antibiotic treatment for shigellosis. The universal emergence of resistant and MDR strains in animals may be attributable to the unrestricted and excessive use of antibiotics in veterinary clinics. The widespread presence of MDR strains has reduced the selectivity of clinical medications to treat shigellosis [30]. Notably, our PFGE dendrogram showed various genetic patterns for S. flexneri, and there were diverse resistance profiles associated with each pattern. Based on these results, S. flexneri has the ability to adapt to the selective pressures of different antibiotics.

The high levels of resistance of S. flexneri 2a to cephalosporin/fluorquinolones, which are the most effective treatments for severe gastrointestinal infections caused by pathogenic bacteria, prompted us to study potential molecular resistance mechanisms. The emergence of ESBL-producing Shigella spp. has been observed in many countries [31]. In the current study, only 3 ARG genotypes (bla OXA-1 , bla TEM-1 and bla CTX-M-14 ) were detected. Among them, the bla TEM-1 gene was detected in all 19 cephalosporin-resistant isolates. In total, 174 bla TEM variants resistant to penicillin and other ß-lactamase antibiotics have been recorded. TEM-1 confers resistance to ampicillin and cephalothin [32]. bla OXA -type ARGs are class D β-lactamases, which were named for their ability to hydrolyze oxacillin [32]. Initially, bla OXA -beta-lactamases were reported in P. aeruginosa, although now the bla OXA gene has been detected in plasmids and integrons in many Gram-negative organisms [32, 33]. According to some studies, the probable host preference for bla OXA -type β-lactamase is S. flexneri [34]. In the present study, 15/19 (78.95%) isolates harbored bla OXA - type genes, and sequencing results indicated that all the bla OXA genes were bla OXA-1 . Additionally, bla CTX-M has become one of the most prevalent extended-spectrum-β-lactamases (ESBLs) [35]. This gene was widely harbored by S. flexneri 2a isolated from beef cattle. Interestingly, all S. flexneri 2a isolated from yaks were negative for bla CTX-M type ARGs.

Fluoroquinolones are highly effective for the treatment of shigellosis worldwide [36]. The primary mechanism of quinolone resistance involves the accumulation of sequential mutations in QRDRs that encode DNA gyrase and topoisomerase IV [37]. The most prevalent mutations in Shigella spp. are the point mutations in gyrA codons 83, 87 and 211, and parC codon 80 [38, 39]. Novel mutations in QRDRs are also being discovered [39]. In the present study, three mutations in gyrA codon 83 (S → L) and/or 87 (D → N or Y) and parC codon 80 (S → I) were detected in each fluoroquinolone-resistant isolate. All substitutions are responsible for reduced affinity. In addition, the amino acid diversity at the same position may lead to different levels of quinolone resistance [40, 41]. GyrA D87Y mutations were detected in only two ciprofloxacin-resistant isolates. However, the role of this mutation in ciprofloxacin resistance is unclear and requires further investigation.

Over the past few years, PMQR determinants have been deemed the most common ARGs in Enterobacteriaceae worldwide [42]. PMQR determinants mediate only low-level quinolone resistance. However, these resistance genes are usually associated with mobile or transposable elements that allow for dissemination among Enterobacteriaceae. In addition, the presence of PMQR genes may facilitate the selection of QRDR mutations that result in higher levels of quinolone resistance [37, 43, 44]. The aac(6)-Ib-cr gene encodes an acetyltransferase that is known to reduce quinolone activity. In the present study, all 14 isolates resistant to fluoroquinolones were positive for aac(6)-Ib-cr, indicating the aac(6)-Ib-cr gene is widespread in S. flexneri 2a. Compared with the aac(6)-Ib-cr gene, the transmembrane segment efflux pump qepA gene was scarcely detected in Shigella, and we found only one ciprofloxacin-resistant isolate that was qepA-positive. The qnr family (which includes the first PMQR genes) contains a variety of subtypes, including qnrA, qnrB, qnrC, qnrD and qnrS and several qnr family genes that have been reported in Shigella [39, 45]. The Qnr proteins protect DNA gyrase against quinolones and facilitate the selection of QRDR mutations that improve resistance to these antimicrobials.

Conclusion

In conclusion, cephalosporin and/or fluoroquinolone resistance in Shigella has been widely reported. To increase our understanding of Shigella in cattle, we investigated Shigella in calves with diarrhea and analyzed the genetic relatedness, antimicrobial susceptibility, QRDR mutations, and prevalence of PMQR and ß-lactamase in S. flexneri 2a isolates from five provinces in northwest China. However, this study also had limitations, including the lack of a systematic surveillance system to prospectively or retrospectively detect and analyze shigellosis in veterinary clinics. Furthermore, we are unable to effectively monitor and control antibiotic abuse and the resulting spread of ARGs. Therefore, it is essential to continually monitor rates of shigellosis and the development of resistance patterns.

Declarations

Acknowledgements

The authors thank all the farms for providing the samples in this study.

Funding

This work was supported by funds from the National Natural Science Foundation of China (31,272,603, 31,101,836).

Availability of data and materials

The data supporting the findings of this study are contained within the manuscript.

Authors’ contributions

Conceived and designed the experiments: JYZ and ZZ. Performed the experiments: ZZ and MZC. Analyzed the data: ZZ, BL, and XZZ. Contributed reagents/materials/analysis tools: MZC and FSC. Wrote the paper: ZZ. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Permission to work in specific locations, information regarding the number of samples harvested, and an associated permit number for calves were not required, and no endangered or protected species were involved or harmed during this study.

Consent for publication

All authors agreed on the publication of the paper.

Competing interests

The authors declare that they have no competing interests.

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Key Laboratory of New Animal Drug Project of Gansu Province, Key Laboratory of Veterinary Pharmaceutical Development of Ministry of Agriculture, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS

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© The Author(s). 2017

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