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Phenotypic identification of Pseudomonas aeruginosa isolated from milk in Taif governorate and characterization of resistance to fluoroquinolones and some medical plant extracts.

INTRODUCTION

Pseudomonas literally means 'false unit', being derived from the Greek pseudo [[TEXT NOT REPRODUCIBLE IN ASCII] 'false'] and monas [TEXT NOT REPRODUCIBLE IN ASCII]. The term "monad" was used in the early history of microbiology to denote single-celled organisms In the year 2000, the complete genome sequence of a Pseudomonas species was determined, recently the sequence of other strains have been examined including Peudomonas aeruginosa strains PAO1 [2000] [1]. The species name aeruginosa was a Latin word meaning copper rust, as seen with the oxidized copper patina on the Statue of Liberty. This also describes the blue-green bacterial pigment seen in laboratory cultures of the species [2]. The ability of Pseudomonas aeruginosa to invade tissues depends on the production of extracellular enzymes and toxins that break down physical barriers and damage most cells, as well as resistance to phagocytosis and the host defense mechanisms. The bacterial capsule or slime layer effectively protects cells from opsonization by antibodies, complement lysis, and phagocyte engulfment. Most Pseudomonas infections were both invasive and toxinogenic. The Pseudomonas infection may be seen as composed of three stages: bacterial attachment and colonization, local invasion and disseminated systemic disease. However, the disease process may supressed at any stage. The bacterial determinants of virulence instance each of these stages and were also responsible for the characteristic syndromes that accompany the disease [3]. Pyocyanin was a virulence factor of the bacteria. However, researchers conclude that salicylic acid could inhibit pyocyanin production. Pseudomonas was also a common cause post-operative infection in radial keratotomy surgery patients. Cystic fibrosis patients were also caused by Pseudomonas aeruginosa infection of the lungs. The organism was also associated with the skin lesion ecthyma gangrenosum [4]. In human, it was the most common cause of infections of burn injuries and of the external ear [otitis externa]. As well as ventilator associated pneumonias, being one of the most common agents isolated in many studies [5]. Pseudomonas aeruginosa infection in animals had been reported as one of the important sources of serious economic losses and was considered all over the world as one of the most dangerous diseases which affects the animal industries also hinders the animal wealth [6]. Pseudomonas aeruginosa had a tendency to cause sudden outbreaks of clinical mastitis in several cows within a few days with no apparent changes in weather and management techniques. Acute cases of mastitis with marked swelling of the udder, high body temperatures and watery milk that contains clots and/or blood. Moreover, infected cows may show severe symptoms of toxemia and some animals died despite continuous treatment. Pseudomonas spp. were naturally resistant to penicillin and all related beta-lactam antibiotics but a few number were sensitive to piperacillin, imipenem and tobramycin [7]. The low susceptibility to most antimicrobial agents was due to a concerted action of multidrug efflux pumps with chromosomally and plasmid encoded antibiotic resistance genes and the low permeability of the bacterial cellular envelopes. Also, intrinsic resistance Pseudomonas aeruginosa easily develop acquired resistance either by mutation in chromosomally encoded genes or by the horizontal gene transfer of antibiotic resistance determinator [1]. Antibiotic resistance might not be associated with the pigment production in Pseudomonas aeruginosa. However pigment production was significantly associated with multi-drug resistance. Presence of virulence-associated genes and expression of certain virulence factors, such as elastase, protease, siderophore and DNase activity. Since pigment production was easy to determine, this might to be an important point to identify the virulence status of an isolate [8]. At present, it was evaluated that about 80% of the world population rely on botanical preparations as medicines to meet their health needs. Herbs and spices were generally considered safe and proved to be effective against certain microorganism's .They were also used in Asian, African and other countries. Nowadays using antimicrobial agents to subside infection was leading to adverse toxicity to host organs, tissues and cells. The toxicity produced by the antimicrobial agents could be prevented or antagonize with herbs [9]. Pseudomonas aeruginosa caused persistent infections in immune compromised individuals and cystic fibrosis sufferers. The infection couldn't cure as P. aeruginosa had developed strong resistance to most conventional antimicrobial agents. The problem was further complicated due to the ability of the pathogen to form biofilm which provides bacterial cells a protected environment withstanding various stresses including antibiotics. Quorum sensing [QS], a cell density--based intercellular communication system, which plays an important role in regulation of the bacterial virulence and biofilm formation, could be a promising target for developing new strategies against P. aeruginosa infections [10]

MATERIAL AND METHODS

Sampling:

A total of 100 samples of different milk samples were examined for Pseudomonas aeruginosa. 11 samples of raw milk from apparently healthy cows, 16 samples of pasteurized cow milk, 14 samples of sterile cow milk, 3 samples of condensed cow milk, 34 samples of raw ewes milk, 5 samples of raw goat milk and 17 samples of raw she-camel milk.

Collection of samples:

For the raw milk samples the udder region, tips of the teats and hands were perfectly washed with water and soap and dried with clean sterile towel then cleaned with 70% ethyl alcohol before the milk sample was taken. The milk was drawn by hand as aseptically as possible, the first four streams being discarded. The samples were collected in clean sterile screw capped bottles [30 ml capacity]. Each sample was labeled to show the animal species, date of sampling and serial number. Collected samples were transferred to the laboratory with a minimum delay. Each sample represented one case and was incubated at 37"C for 24 hrs before cultivation onto solid media.

Cultivation and identification of isolated bacteria:

The samples were cultivated on pseudomonas cetrimide agar, nutrient agar medium and blood agar medium at 37[degrees]C for 24 hours. The suspected colonies were examined for their colonial morphology, hemolytic activity, microscopical examination, and biochemical characters. According to [11], pure cultures were prepared and preserved. Films from the pure suspected colonies were stained by Gram's stain and examined microscopically. These colonies that revealed to be Gram negative, medium sized, straight, non-sporulated bacilli were biochemically examined.

Morphological examination [12]:

Colonial characters onto Pseudomonas cetrimide agar medium: Small greenish colonies were formed with their characteristic features, onto nutrient agar medium: Irregular, round effuse with malt surface, green pigmented colonies were produced and onto blood agar medium: Beta hemolytic, irregular colonies were characterized.

Microscopically examination:

Stained smears from the suspected colonies showed Gram negative rods occurred singly, in pairs or rarely in short chains, none sporulated. One single colony showed typical colonial appearance and morphological characters it was picked up and streaked into semisolid agar medium and incubated at 37[degrees]C for 24 hours for further identification biochemically.

Biochemical identification [11, 13]:

The purified isolates of Pseudomonas aeruginosa were examined by different biochemical reactions Serological identification of Pseudomonas aeruginosa isolates [14]:

Pseudomonas aeruginosa anti-sera [Denka Seiken Co., Ltd. Tokyo-Japan], were used for serological identification of Pseudomonas aeruginosa sero-groups. Test kit was contained 17 monovalent antisera and three polyvalent antisera as follows:

--group A: antibodies to antigens O1,O2,O3,O4 and O5.

--group B: antibodies to antigens O6,O7,O8,O9,O13 and O14.

--group C: antibodies to antigens O10,O11,O12,O15,O16 and O17.

--Normal rabbit serum as negative control.

Susceptibility of isolated strains of Pseudomonas aeruginosa to various antibacterial agents [15]:

Antimicrobial sensitivity discs was done on 15 antimicrobial agents were used:--Amikacin [30ug], Cefotaxime [10ug] Cefepime [30ug], Chloramphenicol [30ug], Ciprofloxacin [5ug], Erythromycin [15ug], Flumequine [30ug] Gentamicin [10ug], Levofloxacin [30ug], Meropenem [10ug], Ofloxacin [5ug], Piperacillin [10ug], Tobramycin [10ug], Tetracycline [30ug], and Trimethoprim sulfamethoxazole[1.25+3.75ug].

Natural plant extract test [16]:

Ethanolic extract is done for the plants in table 4 and the microbicidal activity was classified into resistant if the zone of inhibition in millimeter was less than 8. If it is 9-11mm intermediate, if the inhibition was 12 or more it was sensitive.

Molecular identification of bacterial isolates and detection of resistance gene detection by PCR:

Molecular identification of Pseudomonas aeurginosa isolates were done by detection of groE. PCR was conducted with primers described for the detection of Pseudomonas aeruginosa resistant genes which are ceftazidime related genes [PER & VIM] and also fluoroquinolones related genes [gyrA] [17, 18]. Bacterial genomic DNA extraction was done and Molecular identification of Pseudomonas aeruginosa by Polymerase chain reaction was applied on suspected isolates of Pseudomonas aeruginosa to confirm detection of such pathogens [19]. Molecular detection of antibiotic resistant genes of Pseudomonas aeruginosa ceftazidime resistant [PER & VIM] genes and fluoroquinolones resistance gyrA gen were done [ 17, 18 ], the detection of blaPER and blaVIM, in the ceftazidime resistance isolates and gyrA in the fluoroquinolones resistance isolates was performed by PCR with the specific primers. The PCR amplification was performed in a thermal cycler [Swift MiniPro, ESCO.] by using a 2X DreamTaq DNA PCR master Mix [Thermo Scientific].

Results:

Prevalence of Pseudomonas aeruginosa recovered from different types of milk samples:

Bacteriological examination of a total of [100] different samples obtained from various types of milk, showed that [37] samples harbored Pseudomonas aeruginosa with overall prevalence of [37%] Moreover, it was found that there were variations in the prevalence rate of Pseudomonas aeruginosa isolated from various milk samples. It was higher in raw goat milk [60%], then raw cow milk [54.5%],followed by raw sheep milk [41.2%]. On the other hand, the prevalence rate of Pseudomonas aeruginosa among evaporated cow milk samples was [0%].

Macroscopically examination and direct microscopically examination:

Colonies of Pseudomonas aeruginosa on cetrimide agar medium were large, smooth with flat edges and an elevated appearance producing greenish coloration indicating the presence of pyocyanin exo-pigments. The microscopic examination revealed that 37 isolates appeared as Gram negative, medium sized, straight and non sporulated bacilli.

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Biochemical identification and hemolytic activity test among 37 Pseudomonas aeruginosa isolates:

All Pseudomonas aeruginosa isolates were subjected to different biochemical tests, oxidase test, catalase test, hydrolysis of urea, fermentation of sugar and reduction of nitrate to nitrite. The results reported indicate clearly that all Pseudomonas aeruginosa isolates showed oxidase positive result, urease positive, catalase positive, reduction of nitrate to nitrite, fermentation of glucose, variable fermentation of maltose and did not ferment lactose and negative arginine dehydrolase test. All 37 Pseudomonas aeruginosa isolates had B hemolytic activity.

Results of antimicrobial sensitivity test of Pseudomonas aeruginosa strains:

The in vitro sensitivity tests of Pseudomonas aeruginosa strains from cows, sheeps, goat and camel origin were performed against amikacin, cefepime, cefotaxime, chloramphenicol, ciprofloxacin, erythromycin, flumequine, gentamicin, levofloxacin, meropenem, ofloxacin, piperacillin, tetracycline, tobramycin and Trimethoprim sulfamethoxazole chemotherapeutic agents. Concerning the results of cow milk strains, the most effective antibiotics were gentamicin 85.7%, tetracycline 64.3%, cefotaxime, piperacillin and levofloxacin 50%. These were followed by cefepime and ciprofloxacin42.9%. Amikacin, chloramphenicol, erythromycin, flumequine, tobramycin, ofloxacin, meropenem could be considered in another category and their sensitity percentage ranged between 7.1-35.7%. There was high resistance to trimethoprime sulfamethoxazole reached to 100%. In case of sheep milk strains, the most sensitive antibiotics against Pseudomonas aeruginosa were gentamicin 92.9 %, amikacin 64.3% and piperacillin 57.1 %. Also ciprofloxacin, ofloxacin, and levofloxacin gave sensitivity percentage which ranged from 35.7-50%. Most of the examined strains were highly resistant to cefepime, cefotaxime, tetracycline and erythromycin with a percentage ranged from 78.6-92.9%. In case of camel milk strains, the sensitivity percentage of ciprofloxacin, levofloxacin, ofloxacin, piperacillin and meropenem were [100 %]. Also amikacin, cefepime, chloramphenicol, erythromycin, flomequine, tobramycin and tetracycline gave sensitivity with a percentage of [ 83.3%] . Moreover, cefotaxime and gentamicin gave sensitivity with a percentage of [66.7%]. In case of goat milk strains, the most sensitive antibiotics against Pseudomonas aeruginosa were amikacin, ciprofloxacin, flumequine, gentamicin, tobramycin, piperacillin, meropenem and tetracycline with a percentage of [100%]. Also cefotaxime, cefepime, chloramphenicol, erythromycin, levofloxacin and ofloxacin gave sensitivity with a percentage of [66.7%]. And of high resistance to trimethoprim sulfamethoxazole with a percentage of [66.7%].

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Serogrouping of Pseudomonas aeruginosa isolated from different milk samples:
Sero     Cow    sheep   camel   goat   Total   Percentage
group    milk   milk    milk    milk

O1        5       2       1      0       8       21.62
O2        0       0       1      0       1        2.70
O3        0       3       0      3       6       16.22
O4        5       1       0      0       6       16.22
O5        0       0       2      0       2        5.41
O6        2       1       0      0       3        8.11
O7        0       4       0      0       4       10.81
O8        1       1       0      0       2        5.41
O9        0       0       1      0       1        2.70
O10       1       1       0      0       2        5.41
O11       0       1       1      0       2        5.41
Total     14     14       6      3      37       100 %


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Result of antimicrobial effect of 11 plant extracts against
37 Pseudomonas aeruginosa strains recovered from different
milk samples.

Type of       Isolates    Red    Nigella   Ginger
sample          No.      chili   sativa

Cow milk         14        6        3        6
                           8       11        8
Sheep milk       14        1        5        2
                          13        9        12
Camel milk       6         6        2        6
                           0        4        0
Goat milk        3         1        3        3
                           2        0        0

Type of       Cinnamon   Thyme   Hibiscus   Sensitivity
sample

Cow milk         2         7        1            S
                 12        7        13           R
Sheep milk       3         5        6            S
                 11        9        8            R
Camel milk       6         6        6            S
                 0         0        0            R
Goat milk        2         3        3            S
                 1         0        0            R

Molecular identification of Pseudomonas aeruginosa strains:


Molecular identification of Pseudomonas aeruginosa strains:

All the examined samples showed the same PCR amplicons for groE gene was detected for all Pseudomonas isolates and produced amplicons with average size 536 bp for all samples.

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Result of PCR amplification of ceftazidime resistant blaPER and bla VIM genes:

All the ceftazidime resistance isolates of Pseudomonas aeruginosa were subjected to PCR analyses using specific primers for ceftazidime resistance genes. The results revealed the following: blaPER was detected only in P. aeruginosa resistance isolates of sheep [ 8 out of 12] and cow isolates[6 out of 8] and blaVIM was also detected in P. aeruginosa resistance isolates of sheep [11out of 12] and all cow isolates [8].

The PCR amplicons for blaPER and blaVIM gene were detected and preduced amplicons with average size [925 bp] and [510 bp] respectively

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Electropherotic profile of Pseudomonas aeruginosa blaPER gene of isolates from different sources, Marker 100base pair DNA ladder.

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Electropherotic profile of Pseudomonas aeruginos blaVIM gene of isolates from different sources, Marker 100 base pair DNA ladder

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Results of PCR amplification of the quinolone resistance-determining regions of gyrA gene:

The gyrA gene was successfully amplified from all tested isolates from cow milk and six isolates out of seven from sheep milk. Also, the amplification was succeeded from the reference strains of Pseudomonas aeruginosa ATCC 27853 strains but failed from Pseudomonas aeruginosa strain of goat origin.

The results revealed that, gyrA gene PCR amplicon producted for gyrA gene was of [386 bp] size.

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Electrophoretic profile of PCR products from different Pseudomonas isolates [1-9 cow milk origin] and [10-17 sheep milk origin] amplified by using the primer pairs for gyrA gene. [Marker] 1500 bp, [+] reference strain of P. aeruginosa. [-] control negative Staph. aureus ATCC 29213

Discussion:

Pseudomonas aeruginosa is a Gram negative bacteria which is generally resistant to most of antibacterial agents and considered as one of the microorganism which cause nosocomial infection and one of major problems in many hospitals [21]. Different strains of Pseudomonas aeruginosa, which collected from several sources of milk were the subject of this study. In this study the total number of Pseudomonas aeruginosa isolated from 100 samples of milk from different origins represented as follows 44 cow milk samples, 34 sheep milk samples, 5 goat milk samples and 17 milk samples of camel origins. Positive isolates were 37 with an incidence of 37%, represented as follows: 14 isolates from cow milk samples, 14 isolates from sheep milk samples, 3 isolates from goat milk samples, and 6 isolates from milk samples of camel origins. The recovery rate of raw cow milk, sheep milk, goat milk and camel milk isolates were 54.5 %, 41.2 %, 60 % and 35.3 % respectively. These findings tend to agree with [22] who identified 48 strains of Pseudomonas aeruginosa from milk, respiratory tract, genital tract and wound secretions from diseased cattle suffering from pneumonia, abortion and wound affection in an incidence of 14.3%. Moreover pasteurized cow milk isolates were 25% and sterile cow milk isolates were 28.6% . Also, P. aeruginosa bacteria could not isolated from evaporated cow milk. The results detailed that Pseudomonas aeruginosa organisms were isolated from milk samples of cows with different incidences. Overall incidence reached 54.5 % 6 isolates out of 11 raw cow milk samples. These findings tend to agree with Maaroaf, 2003 [23] who isolated Pseudomonas aeruginosa from clinically mastitic samples and considered it's as a cause of mastitis. Hicks et al., 1991 isolated Pseudomonas aeruginosa from mastitic cows in dairy farm with an incidence of 13.4% [24]. Isolated strains of Pseudomonas aeruginosa from mastitic cow's milk with an incidence ranged from [ 12%-17.6%] [25]. In the present study, Pseudomonas aeruginosa strains were recovered from raw milk samples of sheep. Overall incidence reached 41.2 % 14 isolates out of 34 sheep milk samples. On the other hand, the highest proportion found in goat's milk by 60% 3 isolates out of 5 goat milk samples. This will be dissimilar with Yadav et al., 2014 who did not detect Pseudomonas aeruginosa in goat milk [26]. Pseudomonas aeruginosa isolated from camel milk in this study was 35.3% 6 isolates out of 17 camel milk samples. Many other previous studies have reported the prevalence rates of Pseudomonas aeruginosa in camel which were 1.75%, 1.5%, 1.8%, and 1.66%. It was observed that the rates ranged between 1-1.8% which was different to the rates of our results [27, 28, 29, and 30].

The results of serological typing of P. aeruginosa isolates revealed that the most prevalent serogroupe were serogroupe "O1 " 21.6 % [ 8 isolates] out of 5 isolates from cow milk, 2 isolates from sheep milk and one isolate from camel milk. Serogroupes " O3 and O4 " 16.2 % of each with [ 6 isolates]. The third most frequent serogroupes were serogroupe O7 in a percentage of 10.8% 4 sheep milk isolates . Followed by serogroupe "O6 " 8.1% 3 isolates out of 2 isolates from cow milk and one of sheep milk. Whereas the other serogroups were O5, O8, O10 and O11 2 isolates each in percentage of 5.4%. As regards the low frequeuncy were, serogroupes "O2 and O9 " one isolates for each in a percentage 2.7%. In this concern Wahba., 2005 reported that serotyped Pseudomonas aeruginosa strains isolated from different sources were from type O1, O6 and O8 in respect of their frequencies [31]. Moreover serogroups O1 and O8 were the predominant serogroups among cow's milk [32]. Meanwhile, Pseudomonas aeruginosa serotype O2, O5 and O11 were present in mares suffering from endometritis, vaginal discharges and mastitis [33]. serotypes O2, O3, O4, O6, O9 and O10 were the cause of genital and non-genital infections somatic type O3 accounted for 50 % of isolates [34].

In case of human, the serotype O6 multidrug-resistant strain of Pseudomonas aeruginosa which colonized or infected 201 patients [35]. Also that three most common serotypes present in Spain were found to be O1, O4 and O11 and constituted more than 50% of all isolates [36]. Antibiotics provide the main basis for the therapy of microbial infections. Since the discovery of these antibiotics and their uses as chemotherapeutic agents, there was a belief in the medical fraternity that this would lead to the eventual eradication of infectious diseases. However, over use of antibiotics had become the main cause for the emergence and dissemination of multi-drug resistant strains of several groups of microorganisms [37]. The mechanisms of resistance of Pseudomonas aeruginosa to quinolones occurs by mutation in chromosomal genes [38] through:

Encode the subunits of DNA gyrase and topoisomerase IV [altered target mechanism]:

Regulate the expression of cytoplasmic membrane efflux pumps or proteins that constitute outer membrane diffusion channels [altered permeation mechanism]. In this study, antimicrobial sensitivity test against 15 antibacterial agents revealed that all of the isolates were highly resistant to trimethoprim sulfamethoxazole, cephalothin, cefepime, erythromycin and chloramphenicol. All isolates were highly sensitive to gentamicin and amikacin. In case of cow milk strains, a high resistance were to trimethoprim sulfamethoxazole [100%], tobramycin [92.9%], erythromycin [85.7 %], chloramphenicol [ 78.6 %], amikacin and ofloxacin [71.4 %] and flumequine and meropenem with percentage of [64.3 %] and sensitive to gentamicin [85.7%]. On the other hand, in sheep milk strains, the most resistant to cefepime [92.9%], cefotaxime and tetracycline [85.7%] for each, erythromycin [78.6%], tobramycin, meropenem and trimethoprim sulfamethoxazole with percentage of [ 71.4%] and sensitive to gentamicin [92.9%] and amikacin [64.3%]. Our results confirm that 100% of camel milk strains sensitive to ciprofloxacin, levofloxacin, ofloxacin, piperacillin and meropenem. Also, amikacin, cefepime, chloramphenicol, erythromycin, flumequine, tobramycin and tetracycline gave sensitivity of [83.3%], trimethoprim sulfamethoxazole showed resistance and sensitivity with a percentage of 50%. Moreover, results revealed that the goat milk strains sensitive to amikacin, ciprofloxacin, flumequine, gentamicin, tobramycin, piperacillin, meropenem and tetracycline 100% and resistant to trimethoprim sulfamethoxazole [66.7%] this followed by cefotaxime, cefepime, chloramphenicol, erythromycin, levofloxacin and ofloxacin [33.3%]. all isolates of Pseudomonas aeruginosa were sensitive to gentamicin and neomycin while their resistance to other antibiotics [ampicillin, bacteracin, chloramphenicol, kanamycin, streptomycin and tetracycline] ranged from 50% to 90% [39]. Meanwhile, all isolates were resistant to sulfonamides. 38 isolates of Pseudomonas aeruginosa obtained from six body sites [skin and body orifices] of 53 cows [40]. Most of the isolates were sensitive to colistin, polymyxin-B, and gentamicin. Multiple drug resistance, particularly to ampicillin and oxytetracycline were noticed. On the other hand the susceptibility of 4 strains of Pseudomonas aeruginosa against 9 antibiotics [41]. They detected that, 100% were sensitive to gentamicin, 75% were sensitive to amikacin and polymyxin-B, 100% were resistant to tetracyclines and streptomycin, 75% were resistant to chloramphenicol and sulfa drug and 50% were resistant to erythromycin and kanamycin. Moreover, one hundred strains of Pseudomonas aeruginosa of bovine origin were tested] using different types of antibiotics. They found that, all isolates were sensitive to gentamicin, chloramphenicol, and 90% to kanamycin. In contrast, 100% of the isolates were resistant to ampicillin, streptomycin, 90% to both of tetracycline and erythromycin and 75% to colistin sulphate [42]. Also, 48 isolates of Pseudomonas aeruginosa isolated from cattle, 22 isolates from horses and 21 isolates from various animal species were studied revealed that all strains [100%] were resistant to penicillin, ampicillin, tetracycline and kanamycin and all the isolates were sensitive to gentamicin, polymyxin-B and colistin sulphate[43]. On the other hand the effect of different antibiotics on gentamicin resistant strain of Pseudomonas aeruginosa observed that 95% of isolates were resistant to polymyxin-B while 100% were sensitive to amikacin, 85% to streptomycin, 78% to tetracyclines and 59% to chloramphenicol [44]. Resistance to aminoglycosides was negligible with the exception of streptomycin and kanamycin, only 8% of isolates showed resistance to piperacillin. It was concluded that gentamicin was still very effective against Pseudomonas aeruginosa strains of veterinary origin, irrespective of species and site of isolation[45]. It was also observed that none of the strains were sensitive to chloramphenicol, kanamycin, streptomycin, bactiracin, amikacin, erythromycin, ampicillin, nalidixic acid, nitrofurantoin and penicillin G. On the other hand, antimicrobial surveillance program and documents concerning resistance to a wide range of antimicrobial agents was done. Pseudomonas aeruginosa remained very susceptible to amikacin [91.3-93.8%] > tobramycin > meropenem > piperacillin / tazobactam > gentamicin > piperacillin > cefepime [80.0-81.8%]. While Pseudomonas aeruginosa isolates of Canada were sensitive to aminoglycosides, fluoroquinolones and meropenem [46]. evident from this study that now a day's P. aeruginosa is becoming less sensitive to trimethoprim sulfamethoxazole and cefepime. And cattle milk was somehow contaminated with such antibiotic resistant P. aeruginosa and through the milk systems antibiotic resistance transferred to human and animal. Nowadays using antibiotics to subside infection could be produce adverse toxicity to host organs, tissues and cells. The toxicity produced by the antimicrobial agents could be cured or prevented or antagonized with herbs Herbal molecules were safe, would overcome the resistant produced by the pathogens since they were in combined form. Some herbs said to prevent cancer Some herbs had antibacterial and antifungal properties which would be useful to clinical use [47, 48]. The ethanolic extraction method was revealed that the microbicidal activity of herbs were more than in aqueous extracts [49]. The results showed that ethanolic extracts of Coriander [Coriandrum sativum], Bay leaf [Laurus nobilis], Black pepper [Piper nigrum], Parsley [Petroselinum sativum] and Turmeric [Curcuma longa] did not show any antibacterial activity against 37 Pseudomonas aeruginosa strains with different concentration of each extract [100, 50, and 25%]. While Hibiscus, Thyme [Thymus vulgaris], Black cumin [Nigella sativa], Red chili and Cinnamon [Cinnamomum cassia] gave microbicidal activity against 37 Pseudomonas aeruginosa strains in concentration 100% only with zone diameter ranged between 14-16 mm diameter while in 25and 50% concentration gave no microcidal activity against 37 Pseudomonas aeruginosa strains. The observed resistance of Pseudomonas aeruginosa probably could be due to cell membrane permeability or due to other genetic factors [50]. Concerning the results of cow milk strains, the most effective antimicrobial extract were Thyme with percentage of [50%], Red chili and Ginger with percentage of [42.9%]. These were followed by Nigella sativa, Cinnamon and Hibiscus, could be considered in another category and their effect with percentage 21.4%, 14.3% and 7.1% respectively. In case of sheep milk strains, the most effective antimicrobial extract against Pseudomonas aeruginosa were Hibiscus with percentage [42.9%]. Also Nigella sativa and Thyme gave sensitivity with a percentage of 35.7%. Cinnamon and Ginger gave sensitivity with a percentage of [21.4%] and [14.3%] respectively and of high resistance to Red chili with percentage of [7.1%]. In case of camel milk strains, all tested strains were completely sensitive to Red chili, Ginger, Cinnamon, Thyme and Hibiscus extracts with a percentage of [100%]. And 4 strains had high resistance pattern against Nigella sativa with percentage of [33.3%]. In case of goat milk strains, all tested strains were completely sensitive to Nigella sativa, Ginger, Thyme and Hibiscus extracts with a percentage of [100%]. These were followed by Cinnamon and Red chili extracts gave sensitivity with a percentage of 66.7% and 33.3%. Meanwhile, Hibiscus ethanol extract in concentration of 100 mg\ml gave intermediate antibacterial activity against only seven P. aeruginosa isolated strains with inhibitory zones ranged from 9.4--10.8 mm [51]. These findings were supporting the study results, as Hibiscus gave antibacterial effect for all strains from different types of milk [goat, sheep, camel, and cow]. Moreover, Ginger ethanolic extracts had no activity against Bacillus spp., S. aureus and E. coli [52]. Whereas Ginger ethanolic extracts inhibit the growth of S. aureus and E. coli. However]. Black cumin oil inhibits the growth of S. aureus and P. aeruginosa while E. coli was resistant. Black pepper had antibacterial activity against P. aeruginosa, S. aureus and E. coli. AlsoP. aeruginosa was the most sensitive bacterial strain to Black pepper [53, 54, 55, 56].. The greater resistance of Gram-negative bacteria to plant extracts had been documented previously and was supported by our study [57]. These observations were likely to be the result of the differences in cell wall structure between Gram-positive and Gram-negative bacteria, with the Gram-negative outer membrane acting as a barrier to many environmental and chemical substances, including antibiotics. Herbs could be recommended as antimicrobial agents by conducting further researches. The molecular biology and genetic engineering tools could be utilized to isolate the active principles of herbs and to produce it in large quantities by secondary metabolites synthesis technology. In the issue of molecular identification, all the examined Pseudomonas isolates it showed the same PCR amplicons for groES gene and produced amplicons with average size 536 for all tested samples. According to resistance profile of P. aeruginosa isolates, it was very clear the most resistance groups were P-lactam [ceftazidime], the molecular detection of ceftazidime resistance related genes [blaPER & blaVIM] .There were many studies discussed blaPER & blaVIM genes as a major genes control ceftazidime resistance. The Betalactams PER-1[Pseudomonas extended spectrum beta-lactamase was the first ESBL class A] identified and fully characterized in P. aeruginosa, which occurred in 1993. PER-1 was more prevalent in almost all the countries like Italy, Belgium, France, Spain, Romania, Hungary, Serbia, Korea, Japan, China, Europe, etc. The bla PER-1 gene was found mostly in P. aeruginosa, where it resided within a specific transposon, named Tn1213 and Tn4176 by [58, 59]. The emergence of PER-1 in Gram-negative clinical pathogens possessed a severe challenge to antimicrobial chemotherapy with an increasing number of reports worldwide [60]. In this study, all the ceftazidime resistance isolates of Pseudomonas were subjected to PCR analyses using specific primers for ceftazidime resistance genes. The results revealed that, blaPER was detected only in some of P. aeruginosa resistance isolates of sheep 8 out of 12 and cow origin 6 out of 8 [61, 62]. Second dominant group of acquired MBL was VIM [Verona Integron encoded Metallo P-lactamases] [Veronese Imipenemase] type enzyme, which was first described in Verona, Italy from a Pseudomonas aeruginosa isolate in 1997 which was resistant to piperacillin, ceftazidime, imipenem and aztreonam [63]. The blaVIM-containing integron was located on the chromosome of P. aeruginosa VR-143/97, and the metallo--P-lactamase-encoding determinant. Expression of the integron-borne blaVIM gene in E. coli resulted in a significant decrease in susceptibility to a broad array of P-lactams [ampicillin, carbenicillin, piperacillin, mezlocillin, cefotaxime, cefoxitin, ceftazidime, cefoperazone, cefepime, and carbapenems], revealing a very broad substrate specificity of the VIM-1 enzyme. In this study, blaVIM was also detected in P. aeruginosa resistance isolates of sheep 11out of 12 and all cow isolates 8 and the only one P. aeruginosa resistance isolate of goat milk origin. Quinolone-resistant strains of veterinary origin had also been reported, although there were few data about the resistance mechanisms involved [64, 65]. The results of this study detected, the mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa strains isolated from different source of milk, analyzing the gyrA gene as well as the activity of the efflux pump. The gyrA gene was successfully amplified from all tested isolates from cow milk and six isolates out of seven from sheep milk. Also, the amplification was succeeded from the reference strains of Pseudomonas aeruginosa ATCC 27853 strains but failed from P. aeruginosa strain of goat origin [66].

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(1) Nashwa A. Ezzeldeen, (2) Samar M. Mouneir, (2) Nourah Mohammed Komaikh Al-Zahrani, (1) Afaf Ali Amin El. Haririr, M., (3) Mohamed A. Abdelmonem, (1) Seida A.A. and (3) Manal F AbdelAzez

(1) Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. *Department of Biology, Faculty of science, Taif University.

(2) Department Pharmacology and Toxicology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt., Faculty of Clinical Pharmacy, Taif University

(3) Department of microbiology central lab of residue analysis and pesticides on food

Address For Correspondence:

Mohamed A. Abdelmonem, Department of microbiology central lab of residue analysis and pesticides on food Tel: 00201001471898; E-mail: doctormmm23@hotmail.com

Received 12 February 2016; Accepted 12 March 2016; Available online 22 March 2016
Table 1: the source and type of collected samples.

Type of samples    No. of samples
Raw goat milk            5
Raw sheep milk           34
Raw camel milk           17
Cow milk:
Raw                      11
Pasteurized              16
Sterile                  14
Evaporated               3

TOTAL                   100

Table 2: the main characteristics of the pathogenic
Pseudomonas species [11].

characteristics              P. aeruginosa      P. fluorescens

Pigment produced                  ++                  ++

Odor                      Fruity [grape like]         +
Oxidase test                       +                  +
Growth on MacConkey                +                  +
Growth at 5[degrees]C              -                  +
Growth at 42[degrees]C             +                  -
Oxidation of glucose               +                  +
Oxidation of lactose               -                  -
Arginine dihydrolase               +                  +
Reduction of nitrate               +                  +
  to nitrite
Reduction of nitrate               V                  +
  to N2 gas
Motility                           +                  +

characteristics            P. peudomallei       P. mallei
                                 -                  -
Pigment produced          colonies become     colonies are
                          orange to cream    yellow to brown
                          putrid to earthy

Odor                             +                  -
Oxidase test                     +                  +
Growth on MacConkey              -                  -
Growth at 5[degrees]C            +                  -
Growth at 42[degrees]C           -                  -
Oxidation of glucose             +                  +
Oxidation of lactose             +                  -
Arginine dihydrolase             +                  +
Reduction of nitrate             +                  +
  to nitrite
Reduction of nitrate             +                  -
  to N2 gas
Motility                                            -

+= Positive reaction, [+]= most strains positive,
V= variable reaction,-=negative

Table 3: Interpretation of zones of inhibition in agar
diffusion method for antibacterial susceptibility.

Antimicrobial agent    Symbol   Disc content

Amikacin                 AK     30 [micro]g
Cefotaxime              CTX     30 [micro]g
Cefepime                FEP     30 [micro]g
Chloramphenicol          C      30 [micro]g
Ciprofloxacin           CIP      5 [micro]g
Erythromycin             E      15 [micro]g
Flumequine               UB     30 [micro]g
Gentamicin               GM     10 [micro]g
Tobramycin              TOB     10 [micro]g
Levofloxacin            LEV      5 [micro]g
Ofloxacin               OFX      5 [micro]g
Piperacillin            PRL     100 [micro]g
Meropenem               MEM     10 [micro]g
Tetracycline             T      30 [micro]g
Trimethoprim +          SXT      1.25+3.75
Sulphamethoxazole

                                     Diameter of inhibition

Antimicrobial agent    Resistance    intermediate   sensitive

Amikacin               14 or less       14-16       17 or more
Cefotaxime             14 or less       15-17       18 or more
Cefepime               17 or less       18-22       23 or more
Chloramphenicol        12 or less       13-17       18 or more
Ciprofloxacin          27 or less       28-29       30 or more
Erythromycin           13 or less       14-17       18 or more
Flumequine             13 or less       14-15       16 or more
Gentamicin             12 or less       13-14       15 or more
Tobramycin             15 or less       16-18       19 or more
Levofloxacin           16 or less       17-19       20 or more
Ofloxacin              12 or less       13-15       16 or more
Piperacillin           18 or less       19-21       22 or more
Meropenem              21 or less       22-24       25 or more
Tetracycline           14 or less       15-18       19 or more
Trimethoprim +         10 or less       11-15       16 or more
Sulphamethoxazole

Table 4: natural medicinal plants.

1.Cinnamon [Cinnamomum cassia]                    7.Chilly
2.Coriander [Coriandrum sativum]      8.Parsley [Petroselinum sativum]
3. Turmeric [Curcuma longa]                      9.Hibiscus
4.Ginger [Zingiber officinale]           10.Thyme [Thymus vulgaris]
5. Bay leaf [Laurus nobilis]          11 .Black cumin [Nigella sativa]
6. Black Pepper [Piper nigrum]

Table 5: A list of species specific primer used in
the present study.

Primer Name            Primer Sequence         PCR product   Reference

pseudomonas      5'-ATGAAGCTTCGTCCTCTGCAT-3'
specific
groE heat-          5'-GTCTTTCAGCTCGAT-3'
shock protein

Table 6: A list of antibiotic resistant gene specific
primers used in the present study.

Primer Name            Primer Sequence             PCR     Reference
                                                 product

Extended      PER-F 5'- AATTTGGGCTTAGGGCAGAA-3'   925bp       [18]
spectrum      PER-B 5'- ATGAATGTCATTATAAAAGC-3'
[beta]-       VIM-F 5'- ATGGTGTTTGGTCGCATATC-3'   510bp
lactamases     VIM-B5'- TGGGCCATTCAGCC AGATC-3'
[ESBLs] in
the
ceftazidime
resistance
genes

                               gyrA-F
gyrA              5 ' -AGTCCTATCTCGACTACGCGAT-3 '   386     [17]
                                   gyrA-R
                  5 ' -AGTCGACGGTTTCCTTTTCCAG-3'

Type of samples            No. of samples           Positive samples

                                                    No.   Percentage
Raw goat milk                   5                    3       60%
Raw sheep milk                 34                   14      41.2%
Raw camel milk                 17                    6      35.3%
Cow milk:
Raw                            11                    6      54.5%
Pasteurized                    16                    4      25%
Sterile                        14                    4      28.6%
Evaporated                      3                    0        0%
TOTAL                         100                   37       37%
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Author:Ezzeldeen, Nashwa A.; Mouneir, Samar M.; Al-Zahrani, Nourah Mohammed Komaikh; M, Haririr, Afaf Ali A
Publication:Advances in Environmental Biology
Article Type:Report
Date:Feb 1, 2016
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