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First case of vancomycin resistant Streptococcus alactofyticus from raw milk in Baghdad-Iraq.

INTRODUCTION

Lactic acid bacteria have long been used in industrial applications mainly as starters for food fermentation or as biocontrol agents or as probiotics [1,2]. They form a heterogeneous group of bacteria, the genera of Enterococcus, Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus and Weissella being the best known. Most Lactic acid bacteria are non-pathogenic and they are associated with a wide variety of sources, such as plant material and various foods [3,4].

Group D streptococci, the common inhabitants of the intestinal flora of vertebrates, were first identified by biochemical characteristics and their antigenic [5]. The group D streptococcal strains were then commonly designated as S. equines and S. bovis depending on their biochemical characteristics and on their origin. S. equinus and the type strain of S. bovis belonged to a single species. S. bovis, S. gallolyticus, and S. macedonicus formed a single DNA-cluster including three different subspecies. These were designated as S. gallolyticus subsp. pasteurianus, S. gallolyticus subsp. gallolyticus, and S. gallolyticus subsp. macedonicus. The two other DNA-clusters corresponded to the two subspecies of S. infantarius, and to S. alactolyticus [6]. Group D streptococci have rarely been associated with neonatal infections, Toepfner et al. [7] report a case of fulminant respiratory distress syndrome caused by Streptococcus alactolyticus in a term neonate. Gram staining revealed gram-positive cocci and culture grew group D streptococci in samples taken from ear, trachea and nasopharynx.

Streptococcus alactolyticus was described in 1984 by Farrow and coworkers for a group of streptococci resembling S. equinus, and isolated from faeces of chickens and the intestines of pigs [8]. It is differs from S. equinus by the presence of urease activity. Positive melibiose and mannitol reactions are also indicative for S. gallolyticus. Another species resembling S. alactolyticus is Streptococcus hyointestinalis [9].

Vancomycin has been the cornerstone of treatment of patients with serious Methicillin resistant S.aureus infections. Consequently, vancomycin use has been increasing since the mid-1980's, resulting in the emergence of Methicillin resistant S.aureus with reduced susceptibility to vancomycin [10]. In 1988, Uttley et al. [11] were the first to report the isolation of vancomycin-resistant E. faecium and E. faecalis in England. Shortly after the first isolates of vancomycin-resistant enterococci were reported by investigators in the United Kingdom and France, similar strains were detected in hospitals located in the eastern half of the United States [12].

Various scientific studies support the hypothesis of a link between the use of antibiotics during antimicrobial resistance of human pathogens and agricultural production in which food is one of the possible transfer routes [13,14]. Conjugation in food matrices was reported in experimental studies e.g., the transfer of plasmid-borne ampicillin resistance genes to E. coli K12 from Salmonella Typhimurium in ground beef and inoculated sterilized milk [15], and the transfer of antimicrobial resistance from lactic acid bacteria (Lactococcus lactis, Enterococcus faecalis) to potential pathogenic strains (Staphylococcus aureus, Listeria spp., Salmonella spp., and E. coli) in fermented whole milk (fermented with the Lactic acid bacteria donors)[16]. Since Lactic acid bacteria are usually consumed in a large amount, extrinsic resistance in Lactic acid bacteria, which can be transferred to a normal microbiome, would render a big health problem [17].

There are not reports evaluating S. alactolyticus in Iraq. The objectives of the present study were to isolate vancomycin-resistant S. alactolyticus from raw milk in Baghdad and to clarify the antibiotic resistance in these isolates.

MATERIALS AND METHODS

Milk Samples:

Fifteen raw cow milk's samples were obtained from Baghdad markets. The milk samples were collected in sterile bottles; they were transported to the laboratory in a cool box and stored at 4[degrees]C until analysis.

Isolation and Identification of Bacterial Isolates:

A number of bacterial isolates were isolated from raw cow milk's by the dilution agar method. Briefly, 1 ml of each sample was aseptically transferred into 9 ml of sterile saline solution to make an initial dilution (10-1). Serial dilutions were made for each sample. A volume of 0.1 ml of the dilutions was plated on MRS agar supplemented with 30 [micro]g/ml of vancomycin [18], and incubated in anaerobic conditions at 30[degrees]C for 48 h. Isolated colonies were taken for purification. The purified colonies were tested by microscopic examination with Gram stain, catalase and oxidase production. The Gram positive, cocci shape ,catalase and oxidase- negative isolates were selected for further identification by vitek 2 system.

Minimal Inhibitory Concentrations:

The MICs of vancomycin were determined by a broth dilution method with vancomycin concentrations (2-1024)[micro]g/ml according to the guidelines recommended by the CLSI document.

Antimicrobial susceptibility test:

The vancomycin resistant isolates were subjected to antimicrobial susceptibility testing using Kirby-Bauer disk diffusion method following CLSI guidelines, using commercially available 6mm discs (Bioanalyse /Turkey). The susceptibility of the isolates was determined against 13 antibacterial agents, included: Cefamandol, Cefotaxime, Cloxacillin, Azithromycin, Ampicillin, Ciprofloxacin, Penicillin G, Cephalexin, Colistin, Clindamycin ,mecillinam, tetracycline and Novobiocin.

RESULTS AND DISCUSSION

Twenty seven vancomycin resistant isolates were isolated on MRS vancomycin agar (vancomycin at 30 [micro] g/ml) from 15 raw cow milk samples. The 27 isolates were Gram positive, cocci shape associated in pairs and/or chains, negative catalase and oxidase reaction. After identification by vitek 2, among these 27 isolates, 14 isolates belonged to Streptococcus alactolyticus, 12 isolates Leuconostoc subspecies (Leuconostoc mesenteroides ssp. cremoris (9), Leuconostoc mesenteroides ssp. mesenteroides(2), Leuconostoc mesenteroides ssp.dextranicum(1)), and one isolate belonged to Lactococcus lactis subsp.lactis (Table 1).

Streptococcus alactolyticus is dominating culturable lactic acid bacteria (LAB) species in the jejunal and fecal samples associated with the dogs. In present study, a total of 14 S. alactolyticus isolates have been isolated from raw milk in Baghdad/Iraq.

Streptococcus alactolyticus a Gram-positive cocci, alpha-haemolytic, nonmotile and non-pigmented. Colonies were small, 1 mm in diameter or less. At 42[degrees]C, colony dissociation was seen with some strains having flat or very small colonies, as well as normal slightly heaped-up colonies. No growth occurred in 6.5% NaCl broth [8].

The MIC values for vancomycin in the isolates ranged from 32 to 512 [micro]g/ml (Table 2). Vancomycin is a glycopeptide antibiotic used for the treatment of Gram-positive bacterial infections. Traditionally, it has been used as a drug of last resort.

The resistance patterns of vancomycin resistant S. alactolyticus isolates were determined. All isolates were resistant to cefamandole, ampicillin, cloxacillin, colistin, mecillinam ciprofloxacin and tetracycline ,while all of isolates were sensitive to Azithromycin ,penicillin G and novobiocin (Table 3).

In contrast to our results Gad et al. [19] found that all Lactococcus and Streptococcus isolates from food were susceptible to vancomycin. Salman [20] found that Lactococcus lactis isolated from raw milk were resist to tetracycline, Ampicillin, cephalexin, ceftriaxone, rifampicin, gentamycin, neomycin, streptomycin and trimethoprime, while this bacteria was susceptible to erythromycin and chloramphenicol.

Selective pressure of using antimicrobial agents in both animal and human treatment, and dissemination of antibiotic resistance bacteria has the possibility to aggravate acquisition and spread of genes for antibiotic resistant. Lactic acid bacteria (LAB) are considered to pool the resistant genes and transfer these to pathogenic bacteria [21]. Antimicrobial resistance genes in pathogenic strains and commensal form an indirect risk to public health, as they increase the gene pool from which pathogenic bacteria can pick up resistance traits [22]. The widespread use of antimicrobial agents in human medicine, agriculture and veterinary has undoubtedly accelerated the evolution of pathogenic bacteria, and cause the emergence of strains that have systematically acquired genes for multiple antibiotic resistance [23,24].

Raw food products can be consumed without having undergone prior preservation or processing and therefore hold a substantial risk for antimicrobial resistance transfer to humans, as the eventually present resistant bacteria are not killed. As a consequence, transfer of antibiotic resistance genes between bacteria after ingestion by humans may occur [22].

The biochemical mechanism of vancomycin action is based on the high affinity of vancomycin for the dalanyl-d-alanine residue, a ubiquitous component of the bacterial cell wall precursor Lipid II. The resistance mechanism identified in the Tn1546-based antibiotic resistance was shown to involve alteration of this dipeptide residue from D-ala-D-ala to d- alanyld- lactate, a dipeptide with substantially lower affinity for the antibiotic [25].

The transfer of genes for antimicrobial resistance by conjugation has also been demonstrated in food, namely the transfer of tetracycline resistance genes among lactic acid bacteria in fermented milk [16], among Lactobacillus curvatus in fermented sausages [26], and of vancomycin and tetracycline resistance genes among Enterococcus faecalis during the fermentation process of sausages and cheese [27].

Methods to control unnecessary use of antimicrobial agents include appropriate regulations on use of antimicrobial agents in agriculture, restriction of antimicrobial agents use to pathogen specific agents, and limits on the common practice of using antibiotics for viral infections. Clearly, it is desirable to use antimicrobial agents only when appropriate, to try to limit selective pressure that increases the frequency of antibiotic resistance. Nevertheless, the distinction between causality of bacterial resistance and the rate of spread of antibiotic resistance must be recognized if we are to create a true solution to the antibiotic resistance problem [28].

CONCLUSIONS:

This study had established that vancomycin resistant S. alactolyticus isolates are present in the raw milk in Baghdad. Accurate molecular study for vancomycin resistance in S. alactolyticus requires to try to limit selective pressure that increases the frequency of antibiotic resistance.

ACKNOWLEDGEMENTS

The authors thank Biology Department College of Science, Al- Mustansiriyah University for support this work.

REFERENCES

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Mohammed F. Al Marjani; Jehan Abdul Sattar Salman; Saba Riad Khudhaier; Mustafa Z. Salim and Zahraa A. Kadham

Department of Biology, College of Science Al- Mustansiriyah University, Baghdad, Iraq.

Address For Correspondence:

Mohammed F. Al Marjani, Department of Biology, College of Science Al- Mustansiriyah University, Baghdad, Iraq. E-mail: marjani20012001@yahoo.com

Received 12 February 2016; Accepted 28 March 2016; Available online 25 April 2016
Table 1: Vancomycin resistant cocci isolates from raw milk.

Bacterial Species                               No. of Isolates
Streptococcus alactolyticus                           14
Leuconostoc mesenteroides ssp. cremoris                9
Leuconostoc mesenteroides ssp. mesenteroides           2
Leuconostoc mesenteroides ssp.dextranicum              1
Lactococcus lactis subsp.lactis                        1

Table 2: The MIC values for vancomycin in the S.
alactolyticus isolates.

Bacterial isolate      MIC [micro]g/ml

S. alactolyticus 1     512
S. alactolyticus 2     512
S. alactolyticus 3     32
S. alactolyticus 4     128
S. alactolyticus 5     512
S. alactolyticus 6     512
S. alactolyticus 7     512
S. alactolyticus 8     512
S. alactolyticus 9     512
S. alactolyticus 10    512
S. alactolyticus 11    512
S. alactolyticus 12    512
S. alactolyticus 13    512
S. alactolyticus 14    256

Table 3: The resistance percentage of vancomycin
resistant S. alactolyticus isolates

Antimicrobial agents    Resistance percentage
                        of isolates%

Cefamandol              100
Cefotaxime              92.85
Cloxacillin             100
Azithromycin            0
Ampicillin              100
Ciprofloxacin           100
Penicillin G            0
Cephalexin              42.85
Colistin                100
Clindamycin             42.85
mecillinam              100
Novobiocin              0
Tetracycline            100
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Author:Marjani, Mohammed F. Al; Salman, Jehan Abdul Sattar; Khudhaier, Saba Riad; Salim, Mustafa Z.; Kadham
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAQ
Date:Mar 1, 2016
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