1. Introduction
Acinetobacter species are usually commensal organisms, but they occasionally cause infections, predominantly in susceptible patients in hospitals. They are opportunistic pathogens that may cause urinary tract infections, pneumonia, bacteraemia, secondary meningitis and wound infections. These diseases are predisposed by factors such as malignancy, burns, major surgery and weakened immune systems, in neonates and elderly individuals. There is no evidence of gastrointestinal infection through ingestion of Acinetobacter spp. The emergence and rapid spread of multidrug resistant A. calcoaceticus baumannii complex, causing nosocomial infections, are of concern in health care facilities. A. baumannii is a prevalent species that causes epidemic outbreaks of nosocomial Acinetobacter infections [1] . A. baumannii is occasionally isolated from environmental samples such as soil and water.
The exact natural habitat of many of the Acinetobacter species is yet to be fully understood and may require intense efforts to identify [2] . Thus, overall diversity of habitat, predilection to accumulate antimicrobial resistance, resistant to desiccation, ability to form biofilm, and propensity to cause hospital infection outbreaks make Acinetobacter a remarkable microorganism. A. baumannii strains are generally more resistant than other species of this genus and often express a multi-drugs resistant (MDR) phenotype. Therefore, treatment of nosocomial infections caused by A. baumannii has become complicated because of the widespread antimicrobial resistance among these organisms [3] . The rising trend of resistance in A. baumannii strains, particularly to newer antimicrobial agents, has therefore become a public health care concern. The organism expresses multiple mechanisms of antibiotic resistant that likely leads to the development of multiply resistant or even “pan-resistant” strains.
Some authors have suggested hospital environment as the major reservoir of the multidrug resistant A. baumannii [4] [5] . It still remained apparent that A. baumannii has been isolated from quit a number of other non- hospital sources. For example, Byrne-Bailey et al. in [6] had isolated multidrug resistant A. baumannii in England from soil fertilized with pig manure, and similarly Zhang and his colleagues in [7] had also isolated multi- drugs resistant A. baumannii from livestock in China. Many bacteria were reported as part of the normal intestinal flora of birds. Bird species associating with potentially contaminated environments, such as refuse dumps, sewage treatment facilities, agricultural sites, and bird feeders, are likely to harbor pathogenic bacteria in their intestinal tracts [8] . For example, Craven et al. [9] implied that house sparrows and European starlings may be responsible for transmitting Salmonella spp., Campylobacter jejuni, and Clostridium perfringens to chickens on poultry farms. Ahmed et al. [10] have isolated non-multi-drug resistant A. baumannii from birds’ feces kept in Zoo, Japan. Thus, birds may inadvertently ingest bacteria in their environment, and the bacteria pass through the intestinal tract with no adverse effects to the carrier bird; yet in such cases the birds may aid in dispersal of the bacteria within the environment. The research therefore aims at determining the prevalence of multidrug resistant A. baumannii in birds’ droppings (carrier rate), especially those associated with vegetables that are minimally process and consumed, in Kano State, and thus, accesses the potential role of wild birds in transmission of A. baumannii for public health importance. In Africa, there is paucity of data on the prevalence of A. baumannii carried in the fecal samples of domestics or wild birds feeding in irrigation farms in Nigeria; most data on multidrug resistant A. baumannii were from Europe and Asia. Therefore an attempt to document these in Nigeria will surely complement the efforts made, for epidemiological and clinical forecast.
2. Material and Methods
The study was carried out in 2 irrigation sites, with the first site located along Jakara wastewater canal irrigation farms and the second site located along Sharada canal wastewater irrigation farms, both located in Kano City. All samples were initially processed to separate the non-fermenters from other Gram negative bacilli on MacConkey agar and 5% sheep blood agar at 37˚C for 24 hours. There after identification was done to confirm presence of Acinetobacter spp. Samples were sub cultured from primary isolation media and grown further on nutrient agar (NA), from which colonies on NA were Gram stain, other biochemical tests conducted include oxidase, catalase gelatin liquefaction, motility and other sugar fermentation were undertaken. These were done in accordance with Microbact 24E (Oxoid) for the identification of unknown oxidase negative bacteria.
Species differentiation on Microbact 24E was done on basis of 24 reactions, and inoculates at 37˚C and 44˚C. Antimicrobial susceptibility tests using disc diffusion method was carried out in accordance with [11] using the following antibiotics, Ofloxacin (OFX), Pefloxacin (PEF), Ciproflox (CPX), Amoxicillin-clavulanic acid (AU), Gentamycin (CN), Streptomycin (ST), Ceporex (CEP), Nalidixic acid (NA), Co-trimoxazole (SXT), Ampicilin (PN). The results were interpreted in accordance with Clinical and Laboratory Standards Institute (CLSI) criteria [12] .
Spearman correlation analysis was used to compare the similarities in response to each antibiotic tested on A. baumannii isolated
3. Results
From a total of 48 fecal samples, 24 from each site, 5 (20.83%) Acinetobactaer baumannii isolates were obtained from fecal samples collected along Jakara irrigation farms, while 10 (41.67%) were obtained from fecal samples collected from Sharada irrigation farms. The overall distribution of antibiotic resistant, for Acinetobacter isolates, detected from each site are shown in Table 1. Isolates from all sources were more resistant to ceporex (100.00%) followed by streptomycin with 80.00% and 90.00% resistance phenotype in isolates from Jakara farms and Sharada farms fecal samples respectively. All isolates were less resistant to co-trimoxazole with 20.00% and 50.00% in fecal samples from Jakara and Sharada farms respectively. Multi dug resistant of two or more antibiotics was observed among all isolates, with the highest seen in isolates from fecal sample collected in Jakara, for example number JK16 was resistant to all drugs tested. Impact, forty six (46.67%) of the isolates were resistant to at least 6 antibiotics, as showed in Table 2. Although, the isolates showed considerable multi drugs resistance, there were however few correlation in the patterns of resistant demonstrated by isolates on each antibiotic. For example only three positive values were observed, which were CEP to PN (67.8%, p < 0.01), CPX to
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Table 1. Percentage distribution of Acinetobacter baumannii occurrence in birds’ feces and antibiotic resistant profiles.
Key: No = Total number, % = percentage, ST = Streptomycin, PN = Ampicilin, CEP = Ceporex, OFX = Ofloxacin, NA = Nalidixic acid, PEF = Pefloxacin, CN = Gentamycin, AU = Amoxicillin-clavulanic acid, CPX = Ciproflox, SXT = Co-trimoxazole.
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Table 2. Distribution of Acinetobacter baumannii isolate from birds’ feces and multidrug resistant profiles.
Key: OFX = % = percentage, ST = Streptomycin, PN = Ampicilin, CEP = Ceporex, OFX = Ofloxacin, NA = Nalidixic acid, PEF = Pefloxacin, CN = Gentamycin, AU = Amoxicillin-clavulanic acid, CPX = Ciproflox, SXT = Co-trimoxazole, S = Sensitive, R = Resistant.
ST (57.6%, p < 0.05), AU to SXT and (55.1%, p < 0.05) as shown in Table 3.
4. Discussion
There have been limited data on the occurrence of Acinetobatcer baumannii especially in association with agricultural produce. A. baumannii was reported to multiply not only on human and animal skin, but also in soil and water and thus has a diversity of reservoirs [1] . Our present finding is clearly in support of this hypothesis, as demonstrated by the high occurrence of Acinetobatcer baumannii isolated from birds’ fecal samples. Although other researchers [6] [7] have demonstrated the isolation of Acinetobatcer species from some livestock, there was no report of isolation from wild birds’ fecal sample. Previously, report has demonstrated bacteria as part of the normal intestinal flora of birds, and therefore bird species associating with potentially contaminated environments, such as refuse dumps, sewage treatment facilities, agricultural sites, and bird feeders, are likely to harbor pathogenic bacteria in their intestinal tracts [8] .
The report of Ahmed et al. [10] on isolation of non-multi-drug resistant A. baumannii from birds’ feces kept in Zoo, Japan, does not really demonstrate isolation from wild bird; since the birds were in captivity, this report is more of prevalence or occurrence of A. baumannii in wild birds, whose date were previously scarce for A. baumannii. Previously birds have been implicated in transmission of pathogenic bacteria; for example, Craven et al. [9] implicated house sparrows and European starlings for transmission of Salmonella spp., Campylobacter jejuni, and Clostridium perfringens to chickens on poultry farms. This suggests that wild birds’ fecal samples at farms environment could disseminate multi-drug resistance A. baumannii and the possible risk of contaminating and infecting human population is likely, through the consumption of minimally processed fresh vegetables. This could be supported by previously researches, for example Solomon and his colleagues [13] had demonstrated the ability of E. coli 0157:H7 to enter lettuce plant through the root system and migrate throughout the edible portion of the plant and thus A. baumannii could be expected to have this ability as bacterium; however a more specific research is required to confirm that. Enterobacteriacea were isolated with increasing frequency from fresh produce, including beans, sprouts, cantaloupes, apples, lettuce, [13] and carrot, [14] . In the area of this study Dahiru et al. [15] isolated E. coli Ol57:H7 in cabbage and lettuce leaves; Uzeh and Adepoju [16] had also reported isolation of Escherichia coli O157:H7 and Listeria monocytogenes from different salad vegetables: cucumber, cabbage, carrot, and lettuce. A number of reports on contamination of vegetables and other agents of transmission have been documented in [13] [14] ; as a whole, leafy green vegetables were cited as a source of 26% of the food-borne outbreaks in United States, between 1998 and 1999 [17] .
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Table 3. Antibiotic resistant similarities of isolates to the antibiotics tested.
Key: * = (p) 0.05, ** = (p) 0.01, ST = Streptomycin, PN = Ampicilin, CEP = Ceporex, OFX = Ofloxacin, NA = Nalidixic acid, PEF = Pefloxacin, CN = Gentamycin, AU = Amoxicillin-clavulanic acid, CPX = Ciproflox, SXT = Co-trimoxazole.
While birds scavenging in irrigation farms poses risk of contaminating the environment and even the vegetables with bacteria, A. baumannii remained to be a challenge to public health, especially due to its pronounce multidrug-resistance mechanisms. In this research birds’ fecal samples have demonstrated a high resistance profile to most antibiotics commonly used, not only resistance per say, but a multi-drug or pan drug resistance as demonstrated by some species isolated. Most alarmingly all isolates were resistance to more than one drug, with quite an amount of resistant to at least five drugs. Although this phenomenon was not new in A. baumannii, the isolation from birds’ fecal sample called for close monitoring and surveillance, so as to address the possible occurrence and rapid transmission, of multi-drug resistance phenotype not only within the genus but also among other Enterobacteriaceae in farm environments. The use of fecal materials or dung of goats, sheep, donkey, birds and many other domestic and wild animals as a source of nutrients to crops is well established practice in Nigeria; this habit contributes in directly amplification of the spread of multi-drug resistance A. baumannii and other pathogenic bacteria, even to environments which were never reported to inhabit.
The observation of high resistance by A. baumannii isolated from this work to ceporex (β-lactam drug) and streptomycin (aminoglycoside drug), is in harmony with what was previously reported on Acinetobacter species, which were shown to exhibit different mechanisms of resistance to antibiotics, including the capability to produce enzymes modifying aminoglycosides, β-lactamases of a wide spectrum of activity, carbapenemases, as well as mechanisms resulting from the changes in outer membrane proteins, in penicillin binding proteins and in topoisomerases [18] . These lead to the formation of multidrug resistant (MDR) strains, and even the pandrug- resistant (PDR) strains which are resistant to all available drugs [19] .
The correlation analysis of the resistance phenotype by A. baumannii on the ten antibiotics has demonstrated high positive relationship between ceporex and penicillin (both β-lactam); however cotrimothazole which was more sensitive than other drugs has also demonstrated more than fifty percent positive correlation with amoxicillin-clavulanate. These were in agreement with Bonomo and Szabo, who reported resistance mechanisms that are expressed frequently by Acinetobacter including β-lactamases, alterations in cell-wall channels (poring), and efflux pumps. A. baumannii can become resistant to quinolones through mutations in the genes gyrA and parC and can become resistant to aminoglycosides by expressing aminoglycoside-modifying enzymes [20] , and thus the exhibition of MDR by greater percentage of A. baumannii is isolated on this work. The presence study marks the beginning of understanding the importance of multi-drug resistance phenotype by A. baumannii in agricultural produce. The occurrence or spillover of multi-drug resistance by wild birds suggests a rather new pathway of transmission that may demand further research input, so as to properly suggest best ways and practice in the control and prevention of diseases caused by A. baumannii and other Acinetobacter species.
NOTES
*Corresponding author.