Molecular characterization of antimicrobial resistance in Gram ...

Microbiol Immunol 2011; 55: 318–327 doi:10.1111/j.1348-0421.2011.00323.x

ORIGINAL ARTICLE

Molecular characterization of antimicrobial resistance in Gram-negative bacteria isolated from bovine mastitis in Egypt Ashraf M. Ahmed1 and Tadashi Shimamoto2 1

Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Kafr El-Sheikh University, El-Geish Street, Kafr El-Sheikh 33516, Egypt, and 2 Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima-shi, Hiroshima 739-8528, Japan

ABSTRACT The aim of this study was to characterize the genetic basis of multidrug resistance in Gram-negative bacteria isolated from bovine mastitis cases in Egypt. Multidrug resistance phenotypes were found in 34 of 112 (30.4%) Gram-negative bacterial isolates, which harbored at least one antimicrobial resistance gene. The most prevalent multidrug-resistant (MDR) species were Enterobacter cloacae (8 isolates, 7.1%), Klebsiella pneumoniae (7 isolates, 6.3%), Klebsiella oxytoca (7 isolates, 6.3%), Escherichia coli (5 isolates, 4.5%), and Citrobacter freundii (3 isolates, 2.7%). The most commonly observed resistance phenotypes were against ampicillin (97.0%), streptomycin (94.1%), tetracycline (91.2%), trimethoprim– sulfamethoxazole (88.2%), nalidixic acid (85.3%), and chloramphenicol (76.5%). Class 1 integrons were detected in 28 (25.0%) isolates. The gene cassettes within class 1 integrons included those encoding resistance to trimethoprim (dfrA1, dfrA5, dfrA7, dfrA12, dfrA15, dfrA17 , and dfrA25), aminoglycosides (aadA1, aadA2, aadA5, aadA7, aadA12, aadA22, and aac(3)-Id), chloramphenicol (cmlA), erythromycin (ereA2), and rifampicin (arr-3). Class 2 integrons were identified in 6 isolates (5.4%) with three different profiles. Furthermore, the β-lactamase encoding genes, bla TEM , bla SHV , bla CTX−M , and bla OXA , the plasmid-mediated quinolone resistance genes, qnr and aac(6)-Ib-cr, and the florfenicol resistance gene, floR, were also identified. To the best of our knowledge, the results identified class 2 integrons, qnr and aac(6)-Ib-cr from cases of mastitis for the first time. This is the first report of molecular characterization for antimicrobial resistance in Gram-negative bacteria isolated from bovine mastitis in Africa. Key words antimicrobial resistance, Egypt, Gram-negative, integrons, mastitis

Mastitis is one of the most important diseases in dairy cows throughout the world, and is responsible for significant economic losses to the dairy industry (1). About 150 different pathogens have been associated with bovine mastitis, including a wide range of Gram-negative bacteria (2). The economic impact of clinical mastitis has been

estimated to be about 33–38% of the total health cost for dairy herds (3). Also, mastitis is the most frequent reason for antibiotic use in lactating dairy cattle, given that onethird of the antibiotics given to animals are for mastitis treatment (4); therefore, antimicrobial resistance of mastitis pathogens has received more attention over the past

Correspondence Tadashi Shimamoto, Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima-shi, Hiroshima 739-8528, Japan. Tel/fax: +81 82 424 7897; email: [email protected] Received 15 November 2010; revised 1 February 2011; accepted 6 February 2011. List of Abbreviations: AMC, amoxicillin–clavulanic acid; AMP, ampicillin; ATM, aztreonam; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; CPD, cefpodoxime; CRO, ceftriaxone; CTT, cefotetan; CTX, cefotaxime; FOX, cefoxitin; GEN, gentamicin; IPM, imipenem; KAN, kanamycin; NAL, nalidixic acid; NOR, norfloxacin; OXA, oxacillin; SPX, spectinomycin; STR, streptomycin; SXT, sulfamethoxazole–trimethoprim; TET, tetracycline.

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c 2011 The Societies and Blackwell Publishing Asia Pty Ltd

MDR bacteria from bovine mastitis in Egypt

few years. A routine part of its control involves administration of antibiotics by intramammary and/or systemic routes. Treatment of this disease is also the most common cause of illegal antibacterial residues in milk (5). The presence of mastitis bacteria that are resistant to antimicrobials has obvious implications for the treatment of infected animals. It is of particular concern if the causative agents have multidrug resistance phenotypes. There could potentially also be implications for the transmission of resistance to the consumer if raw, unpasteurized milk or milk products contained such resistant bacteria. The fact that occurrence of antimicrobial resistance varies between countries and regions has the potential to complicate that matter. Food animals and foods of animal origin are traded worldwide; thus, the occurrence of antimicrobial resistance in one country may be a problem for other countries. The genes for resistance traits can be transferred among bacteria of different taxonomic and ecological groups. The increasing occurrence of antibiotic-resistant microorganisms has fueled interest in the genetics and mechanisms of resistance evolved by bacteria to counteract the effect of antimicrobial agents. The fact that resistance genes do not respect phylogenetic, ecological, or geographical boundaries implies that antimicrobial use and the resulting resistance in one ecological niche may have consequences for the resistance situation in another niche (6). The ability of bacteria to acquire and disseminate exogenous genes via mobile genetic elements, such as plasmids and transposons, has been the major factor in the development of multiple drug resistance (6). One of the most important tools for the spreading of antimicrobial resistance among bacteria is a genetic element known as an integron. Integrons are natural genetic engineering systems that incorporate circularized open reading frames, called gene cassettes, and convert them into functional genes (7). The most notable gene cassettes identified within integrons are those conferring resistance to antibiotics (7). Since most integrons are carried on plasmids and transposons, a strong antibiotic selective pressure can potentially result in the mobilization and dissemination of antibiotic resistance genes (7). Of many classes of multi-resistance integrons that have been identified to date, integron classes 1 and 2 are the most frequent in Gram-negative bacteria (8). Resistance of mastitis pathogens to antimicrobial agents is a well-documented challenge in dairy cows. Numerous studies have determined the antibacterial susceptibility patterns of bacteria isolated from mastitis worldwide (9– 13); however, up to now, there have only been a few reports related to the molecular basis of antimicrobial resistance in bacteria isolated from bovine mastitis (14–16). Currently, nothing is known about the molecular basis of antimicrobial resistance in bacteria isolated from cases of mastitis c 2011 The Societies and Blackwell Publishing Asia Pty Ltd

in Africa. Therefore, the present study was performed to characterize the genetic basis of multidrug resistance in Gram-negative bacteria isolated from cases of clinical and sub-clinical bovine mastitis in Egypt.

MATERIALS AND METHODS Sampling, isolation, and identification procedures

A total of 99 milk samples were collected from 86 cows affected with clinical and sub-clinical mastitis. Samples were collected from nine private dairy farms in Egypt suffering from problems of mastitis and a decrease in milk production throughout 2008. All cows with clinical mastitis were examined and sampled (26 milk samples from 26 cows). Clinical mastitis was diagnosed on the basis of the inflammatory reaction and changes in the milk color of the affected quarters. On the other hand, 73 milk samples were collected from 60 cows affected with sub-clinical mastitis. Diagnosis of sub-clinical mastitis was dependent on a positive California Mastitis Test with normal udder and milk color. The samples were aseptically collected from the affected quarter of the cow before any antimicrobial treatment. Prior to sampling, the udder was washed and dried and the teat was disinfected. Approximately 15 ml of the foremilk was discarded and the next 15 ml collected into screw-capped bottles and transferred to the laboratory on ice. All samples were stored at 4◦ C from the time of collection until examination within 3–4 hr. All samples were centrifuged for 15 min at 3000 rpm and a loopful was taken from the sediment and inoculated on MacConkey’s agar. The inoculated plates were then incubated at 37◦ C for 24 and 48 hr. The samples were isolated and identified by conventional techniques (17). Furthermore, all bacterial isolates were confirmed biochemically using the API ´ 20E system (BioMérieux, Marcy-l’Etoile, France). Antimicrobial susceptibility testing

The antimicrobial sensitivity phenotypes of bacterial isolates were determined using a Kirby–Bauer disk diffusion assay according to the standards and interpretive criteria described by the Clinical and Laboratory Standards Institute (18). The following antibiotics were used: ampicillin (AMP), 10 μg; amoxicillin–clavulanic acid (AMC), 20/10 μg; oxacillin (OXA), 1 μg; cefoxitin (FOX), 30 μg; cefotetan (CTT), 30 μg; cefotaxime (CTX), 30 μg; ceftazidime (CAZ), 30 μg; cefpodoxime (CPD), 10 μg; ceftriaxone (CRO), 30 μg; aztreonam (ATM), 30 μg; imipenem (IMP), 10 μg; nalidixic acid (NAL), 30 μg; ciprofloxacin (CIP), 5 μg; norfloxacin (NOR), 10 μg; chloramphenicol (CHL), 30 μg; gentamicin (GEN), 10 μg; kanamycin 319

A.M. Ahmed and T. Shimamoto

(KAN), 30 μg; streptomycin (STR), 10 μg; spectinomycin (SPX), 10 μg; tetracycline (TET), 30 μg; and sulfamethoxazole–trimethoprim (SXT), 23.75/1.25 μg. The disks were purchased from Nissui Pharmaceutical (Tokyo, Japan).

scribed previously (25, 26). Primers for PCR and DNA sequencing are complied in Table 1. A similarity search was also done using the BLAST program.

RESULTS

Bacterial DNA preparation for PCR

Preparation of the bacterial DNA template was as described previously (19). An overnight bacterial culture (200 uL) was mixed with 800 uL of distilled water and boiled for 10 min. The resulting solution was centrifuged and the supernatant used as the DNA template. Amplification reactions were carried out with 10 uL of boiled bacterial suspensions, 250 μM deoxynucleoside triphosphate, 2.5 mM MgCl 2 , 50 pmol of primers, and 1 unit of AmpliTaq Gold DNA Polymerase (Applied Biosystems, Carlsbad, CA, USA). Distilled water was added to bring the final volume to 50 uL. Following PCR, the reaction products were subjected to electrophoresis in a 1.0% (w/v) agarose gel, stained with ethidium bromide and visualized under UV light. Of note, Gram-negative bacteria identified previously by our group and harboring antibiotic resistance genes and class 1 and class 2 integrons were used as positive controls for PCR screening experiments (20). Screening for class 1 and class 2 integrons

The class 1 integron primers, 5 -CS and 3 -CS, which amplify the region between the 5 -conserved segment (5 -CS) and 3 -CS of class 1 integrons, were used as previously described (21). For the detection of class 2 integrons, PCR was performed with the primer pair hep74 and hep51, specific to the conserved regions of class 2 integrons (22). Both DNA strands of the PCR product were sequenced by the dideoxy chain termination method (23) using an ABI automatic DNA sequencer (Model 373; Perkin–Elmer, Waltham, MA, USA). Primers for PCR and DNA sequencing are complied in Table 1. A similarity search was carried out using the BLAST program available at the NCBI BLAST homepage (http://www.ncbi.nlm.nih.gov/BLAST/). Screening for antimicrobial resistance genes

The bacterial isolates were tested for the presence of TEM-, SHV-, OXA-, CTX-M-, and CMY-β-lactamaseencoding genes by PCR using universal primers for the TEM, SHV, OXA, CTX-M, and CMY families, as previously described (20). The florfenicol resistance gene, floR, was detected by using StCM-L and StCM-R primers as previously described (24). Finally, PCR amplification was used for screening of plasmid-mediated quinolone resistance genes, qnrA, qnrB, qnrS and aac(6 )-Ib-cr, as de320

Occurrence of multidrug-resistant Gram-negative bacteria from bovine mastitis

In this study, a total of 112 Gram-negative bacterial isolates were recovered from cases of clinical and sub-clinical mastitis in Egypt (Table 2). Most isolates were from either different mammary quarters of the same cow or from different cows. The bacterial species isolated were Escherichia coli (42 isolates), Klebsiella pneumoniae (19 isolates), Enterobacter cloacae (18 isolates), Klebsiella oxytoca (12 isolates), Citrobacter freundii (10 isolates), Proteus mirabilis (5 isolates), Proteus vulgaris (4 isolates), and single isolates of Pseudomonas stutzeri and Serratia marcescens (Table 2). Testing the antimicrobial susceptibility using disk diffusion tests for the identified bacterial isolates showed that 34 isolates (30.4%), 29 isolates from clinical and 5 isolates from sub-clinical cases, exhibited resistance phenotypes to multiple antimicrobial agents (Tables 2, 3). The multidrug-resistant species were E. cloacae (8 isolates; 7.1%), K. oxytoca (7 isolates; 6.3%), K. pneumoniae (7 isolates; 6.3%), E. coli (5 isolates; 4.5%), C. freundii (3 isolates; 2.7%) and single isolates (0.9%) of P. mirabilis, P. vulgaris, P. stutzeri and S. marcescens (Table 2). The most commonly observed resistance phenotypes were against ampicillin, streptomycin, tetracycline, trimethoprim–sulfamethoxazole, nalidixic acid, chloramphenicol and spectinomycin (Table 3). Occurrence of class 1 and class 2 integrons

PCR and DNA sequencing results identified class 1 integrons in 28 (25.0%) of the Gram-negative isolates (Table 4). The identified gene cassettes within class 1 integrons were as follows: the aminoglycoside adenylyltransferases genes aadA1, aadA2, aadA5, aadA7, aadA12 and aadA22, which confer resistance to streptomycin and spectinomycin; aac(3)-Id, which confers resistance to gentamicin and sisomicin; dihydrofolate reductase genes dfrA1, dfrA5, dfrA7, dfrA12, dfrA15, dfrA17 and dfrA25, which confer resistance to trimethoprim; the chloramphenicol transporter gene cm1A4, which confers resistance to chloramphenicol; erythromycin esterase gene ereA2, which confers resistance to erythromycin; and rifampicin ADP-ribosylating transferase gene arr-3, which confers resistance to rifampicin. Furthermore, PCR screening results detected class 2 integrons in 6 (5.4%) isolates with three different sizes. c 2011 The Societies and Blackwell Publishing Asia Pty Ltd


Table 1. Primers used for PCR and DNA sequencing Sequence (5 to 3 )

Primer Integrons 5 -CS 3 -CS hep74 hep51 β-Lactamases TEM-F TEM-R SHV-F SHV-R SHV-F-2 SHV-R-2 OXA-F OXA-R OXA-F-2 OXA-R-2 CTX-M-F CTX-M-R CTX-M-F2 CTX-M-R2 CMY-F CMY-R Florfenicol StCM-L StCM-R Plasmid-mediated quinolone qnrA-F qnrA-R qnrB-F qnrB-R qnrS-F qnrS-R aac(6 )-Ib-F aac(6 )-Ib-R

Target

Reference

GGCATCCAAGCAGCAAG AAGCAGACTTGACCTGA CGGGATCCCGGACGGCATGCACGATTTGTA GATGCCATCGCAAGTACGAG

Class 1 integron

21

Class 2 integron

22

ATAAAATTCTTGAAGACGAAA GACAGTTACCAATGCTTAATC TT ATCTCCCTGTTAGCCACC GATTTGCTGATTTCGCTCGG CGGCCTTCACTCAAGGATGTA GTGCTGCGGGCCGGATAAC TCAACTTTCAAGATCGCA GTGTGTTTAGAATGGTGA ATTAAGCCCTTTACCAAACCA AAGGGTTGGGCGATTTTGCCA CGCTTTGCGATGTGCAG ACCGCGATATCGTTGGT CCAGAATAAGGAATCCCATG GCCGTCTAAGGCGATAAAC GACAGCCTCTTTCTCCACA TGGAACGAAGGCTACGTA

bla TEM

20

bla SHV

20

whole bla SHV

20

bla OXA

20

whole bla OXA

20

bla CTX−M

20

whole bla CTX−M

20

bla CMY

20

CACGTTGAGCCTCTATATGG ATGCAGAAGTAGAACGCGAC

floR

24

ATTTCTCACGCCAGGATTTG GATCGGCAAAGGTTAGGTCA GATCGTGAAAGCCAGAAAGG ACGATGCCTGGTAGTTGTCC ACGACATTCGTCAACTGCAA TAAATTGGCACCCTGTAGGC TTGCGATGCTCTATGAGTGGCTA CTCGAATGCCTGGCGTGTTT

qnrA

25

qnrB

25

qnrS

25

aac(6 )-Ib-cr

26

Three isolates had class 2 integrons 2.2 kb in size and contained three gene cassettes, dfrA1, sat2 and aadA1; another two isolates had class 2 integrons 2.5 kb in size and contained three gene cassettes, a putative esterase gene, estX,

streptothricin acetyltransferase gene, sat2, which confers resistance to streptothricin, and aadA1. The final isolate contained a class 2 integron 1.2 kb in size associated with two gene cassettes, dfrA1 and sat2 (Table 4).

Table 2. Occurrence of Gram-negative bacteria isolated from clinical and sub-clinical cases of mastitis in Egypt Number of isolates Bacteria Escherichia coli Klebsiella pneumoniae Enterobacter cloacae Klebsiella oxytoca Citrobacter freundii Proteus mirabilis Proteus vulgaris Pseudomonas stutzeri Serratia marcescens Total

Number of multidrug-resistant strains

Clinical mastitis

Sub-clinical mastitis

Total

Clinical mastitis

Sub-clinical mastitis

27 17 16 11 9 5 4 1 1 91

15 2 2 1 1 – – – – 21

42 19 18 12 10 5 4 1 1 112

3 6 7 6 3 1 1 1 1 29

2 1 1 1 – – – – – 5


Total (%) 5 (4.5%) 7 (6.3%) 8 (7.1%) 7 (6.3%) 3 (2.7%) 1 (0.9%) 1 (0.9%) 1 (0.9%) 1 (0.9%) 34 (30.4%)

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Table 3. Results of antimicrobial sensitivity tests for multidrug-resistant Gram-negative bacteria isolated from mastitis Number of resistant isolates Escherichia Klebsiella Enterobacter coli pneumoniae cloacae Antibiotic tested (n = 5) (n = 7) (n = 8) AMP STR TET SXT NAL CHL SPX KAN OXA CPD CIP AMC GEN FOX CTX CPZ CRO ATM CAZ CTT IPM NOR

5 4 5 4 2 3 3 2 – 1 – 2 – 1 – – – – – – – –

7 7 7 7 7 6 6 7 7 5 7 4 5 4 1 3 2 2 4 – – –

8 8 6 8 7 6 8 5 7 4 3 3 4 3 2 3 1 3 1 2 1 –

Klebsiella Citrobacter oxytoca freundii (n = 7) (n = 3) 7 7 7 7 6 7 5 6 5 3 7 4 5 2 3 2 3 – 1 1 – 2

3 3 2 2 3 3 2 2 3 3 2 2 3 3 3 3 3 3 2 2 1 –

Proteus Proteus Pseudomonas Serratia mirabilis vulgaris stutzeri marcescens (n = 1) (n = 1) (n = 1) (n = 1) Total (%) 1 1 1 1 1 – – 1 1 1 – 1 1 1 1 1 – 1 1 1 1 –

– 1 1 1 1 – 1 1 1 – 1 – 1 1 – 1 1 – – – –

1 1 1 – 1 1 – 1 – 1 – 1 – – 1 – 1 – – – 1 –

1 – 1 – – – – – 1 – – – – 1 – 1 – – – – –

33 (97.0%) 32 (94.1%) 31 (91.2%) 30 (88.2%) 29 (85.3%) 26 (76.5%) 25 (73.5%) 24 (70.6%) 24 (70.6%) 20 (58.8%) 19 (55.8%) 18 (52.9%) 18 (52.9%) 15 (44.1%) 13 (38.2%) 12 (35.3%) 12 (35.3%) 10 (29.4%) 9 (26.5%) 6 (17.6%) 4 (11.8%) 2 (5.9%)

AMC, amoxicillin–clavulanic acid; AMP, ampicillin; ATM, aztreonam; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; CPD, cefpodoxime; CRO, ceftriaxone; CTT, cefotetan; CTX, cefotaxime; FOX, cefoxitin; GEN, gentamicin; IPM, imipenem; KAN, kanamycin; NAL, nalidixic acid; NOR, norfloxacin; OXA, oxacillin; SPX, spectinomycin; STR, streptomycin; SXT, sulfamethoxazole–trimethoprim; TET, tetracycline.

Occurrence of antimicrobial resistance genes

PCR and DNA-sequencing identified bla TEM−1 in 23 (20.5%) bacterial isolates (Table 4). The extendedspectrum β-lactamase-encoding genes related to the bla SHV group were identified in 12 isolates; 10 isolates were identified as bla SHV−12 positive; and bla SHV−1 and bla SHV−28 were present in single isolates. The gene bla CMY−2 was identified in four bacterial isolates. Furthermore, the extended-spectrum β-lactamase-encoding gene bla CTX−M−15 was identified in one isolate of K. oxytoca, and bla OXA−30 was identified in two isolates of K. oxytoca and one isolate of E. cloacae. The florfenicol resistance gene, floR, which confers resistance to chloramphenicol and florfenicol was identified in seven (6.3%) bacterial isolates. Furthermore, multiplex PCR screening detected the plasmid-mediated quinolone resistance gene, qnr, in 16 (14.3%) of the tested isolates. DNA sequencing results of the PCR amplicons showed that eight isolates contained the gene qnrS, five isolates had qnrB, and the remaining three were qnrA positive. Also, PCR and DNA sequencing results identi322

fied the recently discovered plasmid-mediated quinolone resistance gene: ciprofloxacin-modifying aminoglycoside acetyltransferase gene aac(6 )-Ib-cr in four (3.6%) isolates.

DISCUSSION Mastitis is the most costly and widespread infectious disease found on dairy farms. Antibiotic treatment is an indispensable tool used by dairy producers to prevent the occurrence of mastitis prior to calving and to cure persistent udder infections at the end of lactation; however, the development of antibiotic-resistant bacteria is of increasing concern. In many instances, the indiscriminate use of antimicrobial agents in the treatment of mastitis in dairy cattle has increased the selection pressure towards antimicrobial resistance (27). Consequently, producers and veterinarians often question the potential risk of drug resistance in dairy cattle. Furthermore, consumers are worried about the use of antibiotics in animal production and their potential effect on the environment and on public c 2011 The Societies and Blackwell Publishing Asia Pty Ltd


Table 4. Resistance phenotype and prevalence of integrons and resistance genes in Gram-negative bacteria isolated from clinical and sub-clinical cases of mastitis Isolate

Bacteria

Source

S31-1

Enterobacter cloacae

Clinical

SP24-2 EM1-1

Enterobacter cloacae Enterobacter cloacae

Clinical Clinical

EM12-1


Clinical

K9


Clinical

48E-1


Sub-clinical

KP2-1


Clinical

KP3-1


Clinical

C4

Klebsiella oxytoca

Clinical

K10

Klebsiella oxytoca

Clinical

EM17-1

Klebsiella oxytoca

Clinical

KP3-2

Klebsiella oxytoca

Clinical

KP6-1

Klebsiella oxytoca

Clinical

39-2

Klebsiella oxytoca

Sub-clinical

40-1

Klebsiella oxytoca

Clinical

S27-2

Klebsiella pneumoniae

Clinical

EM18-1


Clinical

EM14-1


Clinical

25-1


Sub-clinical

KP1-1


Clinical

Resistance phenotypesa AMP, CHL, CIP, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, SPX, STR, SXT AMC, AMP, CHL, CPD, CIP, CPZ, CTT, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, CHL, CPD, CPZ, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, CAZ, CHL, CPD, CPZ, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, CHL, CPD, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, CIP, NAL, OXA, SPX, STR, SXT AMP, ATM, CIP, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, CHL, CIP, CPD, CPZ, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, CIP, CPD, CPZ, CRO, CTX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, CAZ, CHL, CIP, CPD, CRO, CTX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, CIP, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, CIP, GEN, KAN, IPM, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, CHL, CIP, NAL, NOR, STR, SXT, TET AMC, AMP, CHL, CIP, KAN, NOR, STR, SXT, TET AMP, ATM, CAZ, CHL, CIP, CPD, CPZ, CRO, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, CAZ, CHL, CPD, CIP, CPZ, FOX, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, CAZ, CHL, CIP, CPD, CPZ, CRO, CTX, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, CHL, CIP, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, CIP, CPD, FOX, KAN, NAL, OXA, SPX, STR, SXT, TET

Integrons Class 1 (dfrA17-aadA5) Class 2 (estX-sat2-aadA1) Class 1 (aadA2, dfrA15) Class 1 (aadA1)

Other genes – bla TEM−1 bla TEM−1 , qnrS1, aac(6)-Ib-cr

Class 1 (dfrA17-aadA5)

bla TEM−1 , qnrA

Class 1 (dfrA12-orfF-aadA2, aadA1)

bla TEM−1, bla OXA−30 , qnrS1, aac(6)-Ib-cr, floR

Class 1 (aadA1) Class 2 (dfrA1-sat2-aadA1)

–

Class 1 (dfrA15b-cm1A4-aadA2) Class 1 (dfrA12-orfF-aadA2)

bla TEM−1 , floR

Class 1 (dfrA1-aadA1, dfrA15)

bla TEM−1 , bla OXA−30 , aac(6)-Ib-cr


Class 1 (dfrA12-orfF-aadA2, aadA22)

bla TEM−1 , bla CTX−M−15 , bla OXA−30 , qnrS1, aac(6)-Ib-cr bla TEM−1 , bla SHV−12 , qnrS1, floR

Class 1 (dfrA1-aadA1, dfrA25) Class 1 (dfrA12-orfF-aadA2, dfrA25) –

bla TEM−1 , bla SHV−1 , qnrS1, qnrB14 bla TEM−1 , bla SHV−12 , qnrB2 qnrS1

–

bla TEM−1 , bla SHV−12 , qnrB

bla SHV−12 , qnrS1

Class 1 (aadA1)

bla TEM−1 , qnrS1

Class 1 (arr-3-dfrA7, aadA12)

bla TEM−1 , bla CMY−2 , bla SHV−12 , qnrA1, floR bla TEM−1 , bla CMY−2 , bla SHV−12 , qnrA, floR

Class 1 (aadA1, dfrA15)

Class 1 dfrA12-orf-aadA2

bla SHV−12

Class 1 (dfrA12-orfF-aadA2)

bla TEM−1 , qnrB

Continued.


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Table 4. Continued Isolate

Bacteria

Source

KP4-1


Clinical

KP2-2


Clinical

9-2

Escherichia coli

Clinical

16-2

Escherichia coli

Clinical

26-2 35-1 38-1

Escherichia coli Escherichia coli Escherichia coli

Sub-clinical Sub-clinical Clinical

28E

Citrobacter freundii

Clinical

K6


Clinical

K11


Clinical

S19-1

Proteus mirabilis

Clinical

S29-1

Proteus vulgaris

Clinical

36-2 41-1

Serratia marcescens Pseudomonas stutzeri

Clinical Clinical

Resistance phenotypesa AMC, AMP, CAZ, CHL, CIP, FOX, GEN, KAN, NAL, OXA, SPX, STR, SXT, TET AMP, ATM, CHL, CIP, CPD, GEN, KAN, NAL, OXA, STR, SXT, TET AMC, AMP, CHL, KAN, NAL, SPX, STR, SXT, TET AMP, CHL, CPDX, FOX, NAL, STR, TET AMP, NAL, STR, SXT, TET AMP, CHL, SPX, SXT, TET AMC, AMP, KAN, SPX, STR SXT, TET AMC, AMP, ATM, CAZ, CHL, CPD, CPZ, CRO, CTT, CTX, FOX, GEN, NAL, OXA, SPX, STR, SXT, TET AMC, AMP, ATM, CAZ, CHL, CIP, CPD, CPZ, CRO, CTX, FOX, KAN, GEN, IPM, NAL, OXA, SPX, STR AMP, ATM, CHL, CIP, CPD, CPZ, CRO, CTT, CTX, FOX, GEN, KAN, NAL, OXA, STR, SXT, TET AMC, AMP, ATM, CAZ, CPD, CPZ, CTT, CTX, FOX, GEN, IPM, KAN, NAL, OXA, STR, SXT, TET AMC, ATM, CAZ, CPD, CRO, CTX, FOX, NAL, OXA, SPX, STR, SXT, TET AMP, CRO, CTX, TET AMC, AMP, CHL, CPD, CRO, CTX, IPM, KAN, NAL, STR, TET

Integrons

Other genes


bla TEM−1 , bla SHV−12 , qnrB

Class 1 (aac(3)-Id-aadA7)

bla SHV−12

Class 2 (dfrA1-sat2-aadA1)

–

Class 1 (dfrA5-ereA2-aadA1)

–

– Class 1 (dfrA17-aadA5), Class 1 (dfrA17-aadA5),

bla TEM−1 bla TEM−1 bla TEM−1

Class 1 (aadA1)

bla TEM−1 , bla CMY−2

Class 1 (aadA1) Class 2 (dfrA1-sat2)

bla TEM−1 , floR

Class 1 (dfrA15-aadA2, dfrA15) Class 2 (estX-sat2-aadA1)

bla TEM−1 , floR

Class 1 (aac(3)-Id-aadA7, dfrA15)

bla TEM−1 , bla CMY−2

Class 1 (aac(3)-Id-aadA7, dfrA15) Class 2 (dfrA1-sat2-aadA1) – –

–

bla SHV−28 bla SHV−12

AMC, amoxicillin–clavulanic acid; AMP, ampicillin; ATM, aztreonam; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; CPD, cefpodoxime; CRO, ceftriaxone; CTT, cefotetan; CTX, cefotaxime; FOX, cefoxitin; GEN, gentamicin; IPM, imipenem; KAN, kanamycin; NAL, nalidixic acid; NOR, norfloxacin; OXA, oxacillin; SPX, spectinomycin; STR, streptomycin; SXT, sulfamethoxazole–trimethoprim; TET, tetracycline.

health. Bovine mastitis is a multifactorial disease and is one of the most difficult bovine diseases to control (2). In this study, many Gram-negative bacterial isolates were recovered from cases of clinical and sub-clinical mastitis in Egypt (Table 2). The bacterial species isolated were E. coli, K. pneumoniae, E. cloacae, K. oxytoca, C. freundii, P. mirabilis, P. vulgaris, P. stutzeri, and S. marcescens (Table 2). Gram-negative bacteria that commonly cause bovine mastitis are classified as environmental pathogens. Coliforms (lactose-fermenting Gram-negative rods of the family Enterobacteriaceae) are the most common cause of

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this form of mastitis. E. aerogenes, E. coli, K. pneumoniae, and S. marcescens are four common coliform bacteria that cause mastitis (28). The phenomenon of multidrug resistance (MDR) is defined as the ability of a cell to show resistance to a wide variety of structurally and functionally unrelated molecules (29). In this study, 30.4% of Gram-negative isolates were MDR types (Table 2). Ampicillin, streptomycin, tetracycline, trimethoprim–sulfamethoxazole, nalidixic acid, chloramphenicol, and spectinomycin were the most common antibiotics for resistance (Table 3).



The resistance phenotypes of bacteria isolated from bovine mastitis has been reported from many countries; however, the resistance patterns of the bacterial populations vary between countries, which may reflect differences in antimicrobial treatments (9–13). For examples, 90.7% of E. coli isolated from dairy cows with mastitis in USA were MDR types (30); while only 11% of E. coli isolated from clinical bovine mastitis in Finland and Israel were MDR types (31). The great differences observed in antibiotic resistance for bacteria causing mastitis from different countries indicate the importance of antibiotic susceptibility tests and periodic surveillance of the antibiotic susceptibilities of bacteria causing mastitis. Of note, the resistance phenotypes for MDR strains of E. coli isolated in this study (Tables 3, 4) are similar to those reported for MDR E. coli strains identified from diarrheic neonatal calves in Egypt recently (32). That may indicate a possible transmission of these resistant bacteria from the cows to the suckling calves. Furthermore, the resistance phenotypes of Gram-negative bacteria recorded in this study are of great clinical significance because most of the abovementioned antimicrobial agents are among the antibiotics recommended for the treatment of mastitis (27, 33–37). Integrons are mobile DNA elements with the ability to capture genes, mainly antibiotic resistance genes, by sitespecific recombination (7). Integrons play a major role in the spread of antibiotic resistance genes in Gram-negative bacteria. In this study, class 1 integrons were identified in 25.0% of the Gram-negative isolates (Table 4). Class 1 integrons have been reported previously in E. coli strains isolated from bovine mastitis in Switzerland (14), the USA (15), and more recently in China (16). Many of the above-mentioned gene cassettes within class 1 integrons in Table 3 were recently identified in MDR strains of E. coli and Salmonella isolated from neonatal calf diarrhea in Egypt (32, 38) and Salmonella spp. isolated from dairy farms in Japan (19). Class 2 integrons have a structure similar to that of class 1 integrons, but are associated with transposon Tn7 (39). In this study, PCR screening results detected class 2 integrons in 5.4% of the Gram-negative isolates with three different sizes (Table 4). Class 2 integrons containing the classical gene cassettes dfrA1, sat2, and aadA1 were previously identified in E. coli and Salmonella spp. isolated from neonatal calves in Egypt (27, 34). Class 2 integrons containing the unusual gene cassettes estX, sat2, and aadA1 were identified previously from S. enterica serovar Enteritidis isolated from poultry in Japan (19). To the best of our knowledge, this is the first report of class 2 integrons in bacteria isolated from cases of mastitis. Comparison of resistance phenotypes of Gram-negative bacteria and gene cassettes captured by class 1 and class 2 integrons indicates that most of the genes were expressed as phenoc 2011 The Societies and Blackwell Publishing Asia Pty Ltd

types (Table 4.) Therefore, integrons may contribute to the multidrug resistance phenotype of the integron-positive strains, although other resistance mechanisms may be involved in it. Resistance to β-lactam antibiotics in Gram-negative bacteria is primarily mediated by β-lactamases, which hydrolyze the β-lactam ring and inactivate the antibiotic. Many different β-lactamases have been described; however TEM-, SHV-, OXA-, CMY-, and CTX-M-βlactamases are the most predominant in Gram-negative bacteria (40). In this study, bla TEM−1 , a narrow-spectrum β-lactamase gene, which confers resistance to penicillins and first-generation cephalosporins, was identified in 20.5% of the bacterial isolates. Also, the extendedspectrum β-lactamase-encoding genes bla SHV−1 , bla SHV−12 , and bla SHV−28 were identified (Table 4). Furthermore, the ampC β-lactamase-encoding gene bla CMY−2 (which confers resistance to a wide variety of βlactam antibiotics, mainly 7-α-methoxy-cephalosporins, such as cefoxitin and cefotetan) and the extendedspectrum β-lactamase-encoding genes bla CTX−M−15 and bla OXA−30 were also identified (Table 4). The CMY-, CTX-M-, OXA-, SHV-, and TEM-β-lactamases have been detected previously in E. coli and Salmonella spp. isolated from diarrheic neonatal calves in Egypt (32, 38). Of note, the resistance of mastitic pathogens to the extended-spectrum cephalosporins through the production of β-lactamases is a serious clinical problem, as cephalosporins are among the frontline treatment for clinical mastitis (33, 37). To the best of our knowledge, this is the first report of β-lactamases in Gram-negative bacteria isolated from mastitis. floR was identified in 6.3% of bacterial isolates (Table 4). The gene floR has been detected previously in E. coli isolated from cattle in France (41) and from diarrheic neonatal calves in Egypt (32). The identification of the florfenicol resistance gene in bacteria isolated from mastitis could induce treatment failure, as florfenicol could be used as an effective broad-spectrum antibiotic for the treatment of clinical and sub-clinical bovine mastitis (36). Plasmid-mediated quinolone resistance is of great concern since these resistance determinants are potentially spread among bacteria due to plasmid mobility (42, 43). In this study, qnr-related genes (qnrA, qnrB, and qnrS) and aac(6 )-Ib-cr were identified in 14.3% and 3.6% of the tested isolates, respectively (Table 4). The genes qnrB, qnrS, and aac(6 )-Ib-cr were identified previously in E. coli and Salmonella spp. isolated from diarrheic neonatal calves in Egypt (32, 37) and Gram-negative bacteria isolated from zoo animals (20). Also, qnrS was identified previously in S. enterica serovar Typhimurium isolated from animals in Japan (19). To the best of our knowledge, this is the first report of detection and identification of qnr and 325


aac(6 )-Ib-cr from mastitis, and also the first report of qnrA in bacteria of animal origin from Africa. Finally, throughout Table 4, it was clear that most of the antimicrobial resistance phenotypes correlated well with the resistance genes identified in this study. In conclusion, in this study many MDR Gram-negative bacteria were identified from cases of clinical and subclinical mastitis for the first time in Africa. We were also able to genetically analyze these isolates, and various categories of antimicrobial resistance genes were reported for the first time from cases of mastitis. Mastitis remains a disease causing the biggest economic losses to the dairy industry, and the success of mastitis therapy is dependent upon the selection of appropriate antimicrobial therapies. Therefore, periodic surveillance of the antibiotic susceptibilities of pathogenic bacteria isolated from dairy cows with clinical mastitis, as well as molecular characterization of this resistance would be an important measure in detecting emergence and spreading of resistance.

9.

10.

11.

12.

13.

14.

15.

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