Short communication: Genetic characterization of antimicrobial resistance in Acinetobacter isolates recovered from bulk tank milk.

A total of 176 Acinetobacter isolates, including 57 Acinetobacter baumannii originally obtained from 2,287 bulk tank milk (BTM) samples in Korea was investigated for the genetic basis of antimicrobial resistance using molecular methods. In addition, the occurrence and cassette content of integrons were examined and the genetic diversity of A. baumannii strains identified was evaluated. Aminoglycoside-modifying enzyme genes were detected in 15 (88.2%) of the 17 aminoglycoside-resistant Acinetobacter isolates tested. The most common aminoglycoside-modifying enzyme gene identified was adenylyltransferase gene aadB (n = 9), followed by phosphotransferase genes aphA6 (n = 7) and aphA1 (n = 5). Of the 31 isolates resistant to tetracycline, tet(39) was detected in 20 of them. The genetic basis of resistance to sulfonamide was identified in 15 (53.6%) of 28 trimethoprim-sulfamethoxazole-resistant isolates and 9 (32.1%) of them carried both sul1 and sul2 genes. A blaADC-7-like gene was detected in 1 ß-lactam-resistant A. baumannii. Furthermore, class 1 integron was identified in 11 Acinetobacter isolates. Two gene cassettes dfrA15, conferring resistance to trimethoprim, and aadA2, conferring resistance to aminoglycosides, were identified in 8 Acinetobacter isolates. None of the isolates was positive for class 2 or class 3 integrons. Pulsed-field gel electrophoresis revealed that most of the A. baumannii strains from BTM samples were genetically diverse, indicating that the occurrence of A. baumannii strains in BTM was not the result of dissemination of a single clone. Elucidation of resistance mechanisms associated with the resistance phenotype and a better understanding of resistance genes may help in the development of strategies to control infections, such as mastitis, and to prevent further dissemination of antibiotic resistance genes. To the best of our knowledge, this is the first report of molecular characterization of antimicrobial-resistant Acinetobacter spp. from milk.

Prevalence and antimicrobial susceptibility of Acinetobacter from raw bulk tank milk in Korea.

The objective of this study was to determine the prevalence of Acinetobacter spp. in bulk tank milk (BTM) samples from different provinces of Korea and to analyze their antimicrobial susceptibility. Altogether, 2,287 BTM samples were investigated. Among them, Acinetobacter spp. were isolated from 176 BTM samples. Out of 176 Acinetobacter spp., 57 isolates were identified as Acinetobacter baumannii. None of the isolates were resistant to cefepime, imipenem, meropenem, ciprofloxacin, levofloxacin, or colistin. Resistance to amikacin, gentamicin, piperacillin, and cefotaxime was 2.3, 7.4, 2.3, and 4.0%, respectively. Acinetobacter spp. were least susceptible to tetracycline (17.6%), followed by trimethoprim-sulfamethoxazole (15.9%), ceftazidime (10.8%), and ampicillin-sulbactam (10.2%). Overall, A. baumannii strains were susceptible to most of the antimicrobial agents tested compared with other Acinetobacter spp. The Acinetobacter isolates showed 17 different patterns of antimicrobial resistance. The most frequent resistance profile observed was ampicillin-sulbactam (n=13), followed by tetracycline (n=9), ceftazidime-tetracycline (n=8), and trimethoprim-sulfamethoxazole-tetracycline (n=8). The results of this study confirmed that Acinetobacter, including A. baumannii strains, are present in BTM, which clearly showed the importance of examining BTM not only for foodborne pathogens but also for Acinetobacter spp., which could be of public health concern. To the best of our knowledge, this is the first report of Acinetobacter spp. in BTM samples from Korea.

Evaluation of the efficacy of immunocastration vaccine composed of gonadotrophin-releasing hormone conjugated with Salmonella typhimurium flagellin in rats.

Immunocastration is an alternative method to replace surgical castration that is commonly performed in domestic and pet animals. In this study, a new immunocastration vaccine was developed, and its efficacy was evaluated in male rats. Six tandem copies of gonadotrophin-releasing hormone (GnRH) peptide were genetically fused to Salmonella typhimurium flagellin fljB (STF2) that is a ligand of toll-like receptor 5 (TLR5). The recombinant STF2-GnRH protein expressed in Escherichia coli was used as the immunocastration vaccine. Sixteen male rats were equally assigned to four groups. Excluding the control rats, three groups were immunized with 100, 200 and 400 µg of the STF2-GnRH vaccine, respectively. All of the immunized rats developed significantly higher titres of antibodies to GnRH than the control rats. The size and weight of both testes and epididymides from the immunized rats were significantly smaller than those of the control rats. Testicular tissues in the immunized rats demonstrated atrophy of seminiferous tubules and decreased numbers of both spermatogonia and spermatocytes. These data indicate that the newly developed STF2-GnRH vaccine has a potent immunogenicity to GnRH and efficiently suppresses the development of testes in rats.

Antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine mastitis between 2003 and 2008 in Korea.

A total of 1,444 coagulase-negative staphylococci (CNS) isolates from bovine mastitic milk samples collected during 2003-2008 in Korea were identified to the species level. Of 14 species identified, S. simulans, S. haemolyticus, and S. sciuri accounted for over 60% of the isolates. All the CNS isolates were tested for susceptibility to eight antimicrobials commonly used in dairy cattle. With a few exceptions, similar resistance patterns were observed among the CNS species: penicillin and ampicillin showed the lowest activity while amikacin, cephalothin, and gentamycin were highly effective. About 39% (557/1,444) of the CNS isolates were pan-susceptible, while 12% (175/1,444) showed resistance to four or more antimicrobials tested.

Prevalence and antimicrobial susceptibility of gram-negative bacteria isolated from bovine mastitis between 2003 and 2008 in Korea.

The objective of this study was to assess trends in the prevalence and distribution of gram-negative bacteria isolated from bovine mastitis and their antimicrobial susceptibilities during a 6-yr period between 2003 and 2008 in Korea. Escherichia coli, Pseudomonas fluorescens, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter lwoffi/junii, Pseudomonas aeruginosa, and Serratia marcescens were the most commonly observed pathogens during this period. Generally, gram-negative bacteria showed low susceptibilities to most of the antimicrobials tested in this study, except amikacin and gentamicin. Although these 2 aminoglycosides were broadly active against gram-negative bacteria, less than half of those bacteria showed susceptibilities to streptomycin. The beta-lactams, except piperacillin, had the lowest activity among antimicrobials tested in this study. Susceptibilities to chloramphenicol and trimethoprim were fairy high in all genera of gram-negative bacteria, except Acinetobacter spp. and Pseudomonas spp., whereas relatively high resistance to tetracycline was observed uniformly among gram-negative bacteria. There was no significant change in the prevalence of bacterial and the proportion of antimicrobial resistance among gram-negative bacteria isolates during a 6-yr period.

Serotypes of Salmonella isolated from faeces of patients with acute diarrhoea in Gwangju Area, Korea, during 2000-2009.

The purpose of this study was to determine the changing pattern of Salmonella serotypes causing acute diarrhoea in humans in Gwangju area, Korea, during 2000-2009. A total of 596 Salmonella isolated from culture of 29,896 faecal samples of patients with acute diarrhoea were included in this study. Faecal samples were collected from local hospitals and clinics in Gwangju area during January 2000-December 2009. The mean annual frequency of isolates for the 10 years was 2.0% (range, 0.9-6.0). The isolates were serologically classified into 43 different serotypes. The 10 most common serotypes were Salmonella Enteritidis (47.9%), S. Typhimurium (20.4%), S. Braenderup (3.2%), S. Montevideo (2.9%), S. Paratyphi B (2.9%), S. London (2.3%), S. Bardo (1.7%), S. Virchow (1.7%), S. Infantis (1.5%) and S. Typhi (1.5%), accounting for 86% of all the isolates. Temporal variations were observed in the distribution of different Salmonella serotypes over the years, and only S. Enteritidis and S. Typhimurium were persistent throughout the study period. Although age specificity varied with serotypes, Salmonella was isolated most frequently from children below 5 years of age (179/596, 30.0%). A seasonal trend was apparent, and the highest rates were found in the summer months. This is the first report of the annual frequency of isolation of Salmonella serotypes, and seasonal and age-specific patterns of salmonellosis in humans in Gwangju area, Korea, over a decade-long period.

Infectious aetiologies of mastitis on Korean dairy farms during 2008.

The objective of this study was to determine the frequency of different mastitis pathogens in 1255 milk samples collected from 368 lactating cows on 24 dairy farms in Korea during 2008. The proportion of cows and quarter milk samples having SCC200,000 cells/ml, an indicator of udder infection, was 54.3% (200/368 cows) and 35.5% (446/1255 samples), respectively. Of the 446 milk samples subjected to bacteriological examination, 16.5% (74) showed no bacterial growth and 3.5% (16) were contaminated. In total, 356 of 1255 (28.3%) samples were bacteriologically positive, from which 415 bacteria were isolated. The most frequently isolated pathogen was coagulase-negative staphylococci (40.7%), followed by Gram-negative bacteria (19.5%) other than Escherichia coli, Staphylococcus aureus (12.2%), Streptococcus uberis (5.3%), Enterococcus spp. (4.8%), E. coli (4.5%), and environmental streptococci (3.1%) other than S. uberis. This study demonstrates that environmental pathogens were the vast majority of bacteria isolated from mastitic bovine milk samples in Korea.

Antimicrobial resistance of enterococci isolated from mastitic bovine milk samples in Korea.

The aim of this study was to determine the antimicrobial resistance of various species of enterococci isolated from mastitic bovine milk samples. A total of 105 enterococci isolates were examined: Enterococcus faecalis (n = 47), Enterococcus faecium (n = 39), Enterococcus gallinarum (n = 6), Enterococcus avium (n = 6), Enterococcus hirae (n = 5) and Enterococcus durans (n = 2). All the isolates were susceptible to ampicillin, gentamicin and vancomycin, and only a single E. hirae isolate was resistant to ampicillin. In general, the most frequently observed resistance among the enterococcal isolates was to tetracycline (69.5%), followed by penicillin (64.7%), erythromycin (57.1%) and cephalothin (44.7%). A similar antimicrobial resistance pattern was observed among individual species except E. durans, which exhibited only tetracycline resistance. Resistance observed among isolates of E. hirae and E. gallinarum was almost as high as E. faecium and E. faecalis. Of 105 isolates, only six (5.7%) strains of E. faecium were susceptible to all the antimicrobials tested and about 52% (55/105) showed resistance to more than three antimicrobials. The most common multiple resistance pattern was penicillin, tetracycline and erythromycin, which was observed in 32 of 105 (30.4%) isolates. This study demonstrates that enterococcal isolates belonging to minor species showed antimicrobial resistance rates as high as those of E. faecium and E. faecalis, and that monitoring of antimicrobial resistance should not be restricted only to those two major species.

Distribution of streptogramin resistance genes and genetic relatedness among quinupristin/dalfopristin-resistant Enterococcus faecium recovered from pigs and chickens in Korea.

Fifty-four quinupristin/dalfopristin-resistant Enterococcus faecium (QDREF) isolated from chickens and pigs during 2002-2003 in Korea were screened by PCR for the presence of streptogramin resistance genes vatD, vatE, and vgbA, and macrolide resistance gene ermB. None of the QDREF isolates carried vgbA and vatD genes, while vatE and ermB were detected in 9.2% and 74% of the isolates, respectively. Twenty-six percent (14/54) of the QDREF isolates contained none of the resistance determinants tested. Pulsed-field gel electrophoresis (PFGE) patterns revealed high heterogeneity: 47 different patterns for 54 QDREF evaluated. Identical PFGE types were observed in two pairs of chicken isolates and a pair of pig isolates, respectively, but chicken isolates did not share PFGE pattern with pig isolates, suggesting clonal spread of QDREF strain between the same species of animals but not between different species of animals. This is the first report, to our knowledge, of vatE-positive E. faecium isolates and also the first evidence of clonal spread of QDREF strain between animals in Korea.

Using field-based epidemiological methods to investigate FMD outbreaks: an example from the 2002 outbreak in Korea.

Relevant to foot and mouth disease (FMD), most published epidemiological studies have been conducted using quantitative methods and substantial regional or national datasets. Veterinary epidemiology also plays a critical role during outbreak investigations, both to assist with herd-level decision-making and to contribute relevant information to assist with ongoing national or regional control strategies. Despite the importance of this role, however, little information has been published on the use of applied (field-based) epidemiological methods during disease outbreaks. In this study, we outline an investigative template for FMD, and a case study of its use during the 2002 FMD outbreak in Korea. Suitable for use during field-based epidemiological investigations of individual farms within a broader regional/national response, the template considers three steps including confirming infection, estimating date of introduction and determining method of introduction. A case study was conducted on IP13 (the 13th infected premises), the only IP during the 2002 FMD outbreak in Korea that was geographically isolated from all other known cases. The authorities first became aware of FMD on IP13 on 2 June, however, infection may have been present from 12 May. Infection was confirmed on 3 June 2002. FMD was probably spread to IP13 by a contract worker who had participated during 2-4 May in the culling operations on IP1. Other routes of spread were ruled out during the investigation. The contract worker lived in the locality of IP13 and worked on a part-time basis at a pork-processing plant that was adjacent to this farm. The contractor became heavily contaminated during the cull, but did not comply fully with cleaning and disinfection requirements once the cull had been completed. The investigative template contributed structure and focus to the field-based investigation. Results from this case study demonstrate the need for strict management of personnel in disease control and adherence to the sanitary rules by all those involved.

Epidemiological characteristics of the 2002 outbreak of foot-and-mouth disease in the Republic of Korea.

The Republic of Korea experienced a foot-and-mouth disease (FMD) outbreak during May-June 2002. The present study describes epidemiological characteristics of the 2002 FMD outbreak in Korea, including the pattern of the outbreak in both time and space, transmission routes among infected farms, and control measures. One of the notable features of the 2002 FMD epidemic in Korea was that the virus infected mostly pigs [15 of 16 infected premises (IPs)], despite the presence of other susceptible animals on infected and neighbouring farms. The epidemic showed temporal clustering at 8-9 day intervals, suggesting five generations of infection during the outbreak, and 13 of 16 (81.3%) IPs were located within a 10 km-radius of the index case. The clinical signs that prompted notification of infection included vesicles around hooves and snouts. The age of lesions was significantly less among cases reported by farmers compared with veterinarians. The high awareness of farmers from an earlier FMD outbreak greatly helped the animal hygiene authority in efforts associated with disease control and eradication. The outbreak was eradicated within <2 months as a result of the intensive control efforts of the animal hygiene authorities and the cooperation of the Korean people. Although the outbreak was a costly lesson for the Korean people, the experience gained will contribute to future efforts in the prevention and control of animal infectious diseases.

Rabies, an emerging disease in Korea.

Since the first rabies case was reported in a dog in 1907, the disease was enzootic up to 1975 in Korea. After a steady decrease in the number of rabies cases from 1976 to 1984, no case was reported for 8 years from 1985 to 1992. Then, a resurgence of the disease was noted in 1993, and a continuous increase of rabies cases was observed during the following years. This report provides information on rabies in South Korea during the reemerging period 1993-2003. A total of 364 rabies cases in five different animal species and five deaths in human beings as a result of rabies were reported. Cattle and dogs accounted for 46.4% and 40.4% of total animal cases, respectively, and raccoon dogs commanded an overwhelming majority (44/48) of rabies cases in wildlife animal species. All animal and human rabies cases occurred only in two provinces, Gyeonggi and Gangwon; majority of them in two counties of Gyeonggi and one county of Gangwon province that border the demilitarized zone. From the three counties, the disease continued to expand to the other areas of the two provinces. The average monthly frequency of animal rabies cases during the 11-year period peaked in January, and the incidence was highest during winter. There were three major rabies outbreaks in animals and the number of animal rabies cases increased with time. Data indicate that the temporal patterns were attributable to the ethology of raccoon dogs in the areas of outbreak.

Virus spread pattern within infected chicken farms using regression model: the 2003-2004 HPAI epidemic in the Republic of Korea.

During the 2003-2004 epidemic in Korea, the infection time and within-farm spread pattern of virus were analysed for the highly pathogenic avian influenza (HPAI) outbreak on chicken farms using regression models based on epidemiological data. Mortality observed on a given day had a positive linear association with time after initial infection. HPAI spread more rapidly on farms managed by employees and on farms with larger numbers of chicken houses in use. The disease spread more rapidly among layer chickens than among broilers. Using statistical model, we found that farmers recognize the abnormally high mortality resulting from HPAI approximately 5 days after infection. Without any intervention, entire flocks would die within 12 days of introduction of the HPAI virus to the infected farm.

Detection of Campylobacter jejuni in dairy farm environmental samples using SYBR Green real-time polymerase chain reaction.

The aim of this study was to evaluate a SYBR Green based real-time PCR assay using well-characterized primers to detect Campylobacter jejuni in naturally contaminated dairy farm environmental samples. Specificity of the assay was determined with 62 C. jejuni strains and 120 non-C. jejuni strains. Peak melting temperature obtained with melting curves specific for C. jejuni was 77.5 degrees C. Standard curves were constructed using mean threshold cycle (C(T)) and various concentrations of C. jejuni ranging from 10(0) to 10(8) colony forming units (CFU)/mL, which resulted in a linear relationship between C(T) and log input DNA. Correlation coefficients of standard curves based on pure culture of C. jejuni in broth and spiked cells in lagoon water were R(2) = 0.995 (slope = 3.21) and R(2) = 0.988 (slope = 3.22), respectively, and sensitivity limits were <10 and >10(3) CFU/mL, respectively. After 24-h enrichment, total C. jejuni counts of all samples spiked with 10(0) CFU/mL reached >10(5) CFU/mL, and the detection limit was improved from >10(3) CFU/mL to <10 CFU/mL of inoculum in broth. Eighty-two dairy farm environmental samples, including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding material, were analyzed. The real-time PCR assay detected C. jejuni in 25 (30.4%) of 82 samples, with 17 (68%) of these samples being culture positive for C. jejuni. All samples that were positive by standard culture methods were also positive by the real-time PCR method. Mean C( T ) values of 48-h enriched cultures for 17 PCR-positive/culture-positive samples and eight PCR-positive/culture-negative samples were 21.4 +/- 3.6, and 34.6 +/- 1.5 (p < 0.0001), respectively. C( T ) values for negative samples were >38.0. These results indicate that the SYBR Green real-time PCR assay provides a specific, reproducible, and simple method for detecting C. jejuni in dairy farm environmental samples.

Prevalence of antimicrobial resistance genes in Listeria monocytogenes isolated from dairy farms.

Antimicrobial resistance of Listeria monocytogenes (n = 38) isolated from the four dairy farms to 15 antimicrobial agents was evaluated. All 38 L. monocytogenes isolates from the four farms evaluated were resistant to more than one antimicrobial in different combinations. All L. monocytogenes isolates evaluated were resistant to cephalosporin C (minimum inhibitory concentration [MIC] > or = 512 microg/mL), streptomycin (MIC > or = 32) and trimethoprim (MIC > or = 512). Most L. monocytogenes isolates were resistant to ampicillin (92%, MIC > or = 2), rifampicin (84%, MIC > or = 4), rifamycin (84%, MIC > or = 4), and florfenicol (66%, MIC > or = 32) and some were resistant to tetracycline (45%, MIC > or = 16), penicillin G (40%, MIC > or = 2) and chloramphenicol (32%, MIC > or = 32). All L. monocytogenes isolates were susceptible to amoxicillin, erythromycin, gentamicin, kanamycin and vancomycin. Susceptibility of L. monocytogenes to the antimicrobials evaluated was quite consistent among the dairy farms evaluated. However, some variability in antimicrobial susceptibility among dairy farms was noted. Nineteen of 38 L. monocytogenes isolates contained more than one antimicrobial resistance gene sequence. A high frequency of floR (66%) was found in L. monocytogenes followed by penA (37%), strA (34%), tetA (32%), and sulI (16%). Other tetracycline resistance genes (tetB, tetC, tetD, tetE, and tetG) and other antimicrobial resistance genes (cmlA, strB, aadA, sulI, vanA, vanB, ampC, ermB, ereA, and ereB) were not found in any of the L. monocytogenes isolates from the four dairy farms. Results of the present study demonstrated that L. monocytogenes isolated from the dairy farm environment were resistant to many antimicrobials and contained one or more antimicrobial resistance genes.

Evaluation of universal pre-enrichment broth for isolation of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples.

Use of universal pre-enrichment broth (UPB) as a primary enrichment medium for detection of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes from dairy farm environmental samples was evaluated. There were no differences in bacterial growth between UPB and selective primary enrichment broths for each pathogen inoculated individually or in combination at 10(1) and 10(2) colony forming units/mL. In addition, no differences were observed when UPB and selective primary enrichment broths were compared for detection efficiency of pathogens in artificially contaminated raw milk and fecal samples. Listeria enrichment broth (LEB) was compared with UPB to support growth of L. monocytogenes from naturally contaminated environmental samples. Listeria monocytogenes was isolated from seven of 30 samples enriched in UPB and six of 30 samples enriched in LEB. Dairy farm environmental samples were examined for recovery of the three pathogens using UPB. Subsequent isolation was achieved using selective secondary enrichment of each pathogen. Listeria monocytogenes, Salmonella spp., and E. coli O157:H7 were isolated in 13.4% (30 of 224), 8.9% (20 of 224), and 2.2% (five of 224) of samples, respectively. Isolation rates of the three pathogens were somewhat higher than in previous reports. Overall, UPB supported growth of test pathogens to detectable levels within 24 h. Our results demonstrate that UPB has potential for routine use in isolation of foodborne pathogens from diverse environmental samples.

Detection of sorbitol-negative and sorbitol-positive Shiga toxin-producing Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, and Salmonella spp. in dairy farm environmental samples.

Six visits were conducted to four dairy farms to collect swab, liquid, and solid dairy farm environmental samples (165 to 180/farm; 15 sample types). The objective of the study was to determine on-farm sources of Campylobacter jejuni, Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC), which might serve as reservoirs for transmission of pathogens. Samples were analyzed using mostly U.S. Food and Drug Administration's Bacteriological Analytical Manual protocols; however, Salmonella spp., L. monocytogenes and STEC were co-enriched in universal pre-enrichment broth. Campylobacter jejuni were enriched in Bolton broth containing Bolton broth supplement. Pathogens were isolated on agar media, typed biochemically, and confirmed using multiplex polymerase chain reaction protocols. Campylobacter jejuni, Salmonella spp., L. monocytogenes, Sorbitol-negative (SN)-STEC O157:H7, and sorbitol-positive (SP)-STEC, respectively, were isolated from 5.06%, 3.76%, 6.51%, 0.72%, and 17.3% of samples evaluated. Whereas other pathogens were isolated from all four farms, SN-STEC O157:H7 were isolated from only two farms. Diverse serotypes of SP-STEC including O157:H7, O26:H11, O111, and O103 were isolated. None of the five pathogen groups studied were isolated from bulk tank milk (BTM). Most pathogens (44.2%) were isolated directly from fecal samples. Bovine fecal samples, lagoon water, bedding, bird droppings, and rat intestinal contents constituted areas of major concern on dairy farms. Although in-line milk filters from two farms tested positive for Salmonella or L. monocytogenes, none of the pathogens were detected in the corresponding BTM samples. Good manure management practices, including control of feral animals, are critical in assuring dairy farm hygiene. Identification of on-farm pathogen reservoirs could aid with implementation of farm-specific pathogen reduction programs.