Heavy metal resistance in bacteria from animals.

Resistance to metals and antimicrobials is a natural phenomenon that existed long before humans started to use these products for veterinary and human medicine. Bacteria carry diverse metal resistance genes, often harboured alongside antimicrobial resistance genes on plasmids or other mobile genetic elements. In this review we summarize the current knowledge about metal resistance genes in bacteria and we discuss their current use in the animal husbandry.

Selection and transfer of an IncI1-tet(A) plasmid of Escherichia coli in an ex vivo model of the porcine caecum at doxycycline concentrations caused by crosscontaminated feed.

AIMS: The aim of this study was to investigate the effect of subtherapeutic intestinal doxycycline (DOX) concentrations (4 and 1 mg l-1 ), caused by cross-contamination of feed, on the enrichment of a DOX-resistant commensal Escherichia coli and its resistance plasmid in an ex vivo model of the porcine caecum. METHODS AND RESULTS: A DOX-resistant, tet(A)-carrying, porcine commensal E. coli strain (EC 682) was cultivated for 6 days in the porcine caecum model under different conditions (0, 1 and 4 mg l-1 DOX). EC 682, other coliforms and anaerobic bacteria were enumerated daily. A selection of isolated DOX-resistant coliforms (n = 454) was characterized by rep-PCR clustering, PCR assays (Inc1 and tet(A)) and micro broth dilution susceptibility tests (Sensititre). Both 1 and 4 mg l-1 DOX-enriched medium had a significantly higher selective effect on EC 682 and other resistant coliforms than medium without DOX. Transconjugants of EC 682 were isolated more frequently in the presence of 1 and 4 mg l-1 DOX compared to medium without DOX. CONCLUSIONS: Subtherapeutic intestinal DOX concentrations have the potential to select for DOX-resistant E. coli, and promote the selection of transconjugants in a porcine caecum model. SIGNIFICANCE AND IMPACT OF THE STUDY: Cross-contamination of feed with antimicrobials such as DOX likely promotes the spread of antimicrobial resistance. Therefore, it is important to develop or fine-tune guidelines for the safe use of antimicrobials in animal feed and its storage.

Nosocomial Intravascular Catheter Infections with Extended-spectrum Beta-lactamase-producing Escherichia coli in Calves after Strain Introduction from a Commercial Herd.

An outbreak of intravascular catheter-related infections by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in calves in an animal teaching hospital is reported. Pulsed-field gel electrophoresis was used for strain typing to determine the origin and dissemination of these strains. All 19 strains harboured the blaCTX-M-14, and six strains also overexpressed their chromosomal AmpC gene. Evidence on the introduction of the strain from a beef herd, experiencing neonatal diarrhoea and increased mortality, to the clinic through admission of diarrhoeic calves was provided. Strains isolated from phlebitis cases from other herds up to 5 months later showed a high similarity with the initial strain, suggesting that the strain had become nosocomial. The catheter infections with ESBL/AmpC-producing E. coli resulted in a prolonged hospitalization, increased anti-microbial use and mortality. This report points towards the potential dangers of the emergence of ESBL/AmpC-producing bacteria in susceptible food animals and warns farmers and veterinarians for the facility by which they are introduced into another environment.

Use of a live attenuated Salmonella enterica serovar Typhimurium vaccine on farrow-to-finish pig farms.

Salmonella enterica infection in pigs is economically important and poses a zoonotic risk. In this study, the efficacy of an attenuated S. enterica serovar Typhimurium strain was evaluated in three farrow-to-finish pig herds. In each herd, 120 piglets were vaccinated orally at 3 and 24 days of age, while 120 piglets served as unvaccinated controls. Faeces, ileocaecal lymph nodes and caecal contents were examined for S. Typhimurium by isolation and serum was analysed for antibodies against S. Typhimurium by ELISA. All pigs were weighed at pre-weaning and slaughter to determine daily weight gain. In vaccinated pigs prior to slaughter, significantly fewer animals excreted S. enterica, there was a significantly lower S. enterica-specific mean antibody titre and there was a significantly higher mean daily weight gain compared to unvaccinated controls. In two herds, there were significantly lower proportions of S. enterica positive ileocaecal lymph nodes and caecal contents at slaughter between the vaccinated and control groups, but this difference was not significant across all three herds. S. enterica with the same auxotrophic characteristics and genotype as the vaccine strain was isolated from several samples of faeces, ileocaecal lymph nodes and caecal contents from vaccinated pigs. These findings indicate that vaccination with an attenuated S. Typhimurium strain reduces S. enterica shedding, but the reduction is not consistent and the vaccine strain may persist in tissues.

Identification of a novel plasmid-associated spectinomycin adenyltransferase gene spd in methicillin-resistant Staphylococcus aureus ST398 isolated from animal and human sources.

OBJECTIVES: Previously described methicillin-resistant Staphylococcus aureus (MRSA) ST398 strains revealed a high frequency of phenotypic resistance to spectinomycin. However, only a few were found to carry the spc resistance determinant. The aim of this study was to identify the genetic mechanism of spectinomycin resistance among spc-negative MRSA ST398 strains. METHODS: Nine spectinomycin-resistant, but spc-negative, MRSA ST398 strains were analysed. The strains were screened for carriage of the spw gene and tested for the presence of transferrable spectinomycin resistance. Plasmid DNA was isolated from all strains and used in transformation assays. The plasmid identified as mediating resistance to spectinomycin was fully sequenced. The function of the novel spectinomycin resistance gene was confirmed by restriction digest inactivation and its distribution was determined using a PCR assay. RESULTS: A single MRSA ST398 strain was spw positive. The remaining strains carried a plasmid that mediated resistance to spectinomycin. Sequence analysis of a single plasmid, termed pDJ91S, revealed that it was 3928 bp in size and contained three open reading frames: a novel spectinomycin resistance gene, designated spd, as well as a repN gene and a rec gene. The XmnI digest inactivation of the spd gene resulted in a 4-fold decrease in spectinomycin MIC. The spd gene was detected in seven other spectinomycin-resistant MRSA ST398 strains that carried a plasmid comparable in size to pDJ91S. CONCLUSIONS: A novel gene, designated spd, that confers resistance to spectinomycin has been identified on a small plasmid in MRSA ST398.

High seroprevalence of respiratory pathogens in hobby poultry.

Seroprevalence studies on respiratory pathogens have been done extensively in commercial laying hens, broilers, and, to a lesser extent, backyard poultry. In Europe, seroprevalence studies in backyard and fancy breed poultry flocks are scarce and limited to a few pathogens, such as Mycoplasma gallisepticum (MG); others, such as Ornithobacterium rhinotracheale (ORT), are missing. A commercial ELISA for detection of antibodies against six selected pathogens was performed on 460 serum samples from chickens across Flanders. Anti-ORT antibodies were, by far, the most prevalent, with a prevalence of 95.4%. Infectious bronchitis virus, Mycoplasma synoviae, and avian metapneumovirus antibodies were found in 75.6%, 76.3%, and 63.5% of the animals, respectively. Antibodies against MG and infectious laryngotracheitis virus were found in 36.7% and 30% of the animals, respectively. These data demonstrate the high seroprevalence of respiratory pathogens among hobby poultry; therefore, it is possible that this group could act as a reservoir for commercially kept poultry.

Enteropathogens in pups from pet shops and breeding facilities.

OBJECTIVES: To evaluate faecal and clinical scores and presence of several enteropathogens possibly implicated in the development of diarrhoea in pups aged between 6 and 16 weeks independently of their health status. METHODS: Pups were selected from pet shops and breeding facilities and assigned a faecal and clinical score. Standard isolation methods were used to determine presence of parasites, viruses and bacteria in faecal samples. For Escherichia coli, virulence genes were assessed by multiplex polymerase chain reaction. RESULTS: Fifty-six pups were included in this study. Eighteen had no diarrhoea, 22 had no significant clinical signs related to gastroenteritis. Samples were positive for Toxocara canis (n=29), Giardia duodenalis (n=35), Cystoisospora (n=22), E. coli (n=47) and Clostridium perfringens (n=20). In four E. coli positive samples, genes were detected that correlate with pathogenicity in other animal species. A significant positive correlation between the presence of T. canis and faecal score was found. CLINICAL SIGNIFICANCE: Puppies obtained from a pet shop or breeding facility have a high risk of gastrointestinal disease. Furthermore, infectious agents may be present independently of faecal or clinical score. The identification of possible pathogenic E. coli strains suggests that their role in diarrhoea warrant further investigation.

Evaluation of three intervention strategies to reduce the transmission of Salmonella Typhimurium in pigs.

Despite current control measures, Salmonella in pigs remains a major public health concern. In this in vivo study, the effect of three intervention strategies on Salmonella Typhimurium transmission in pigs was evaluated. The first intervention was feed supplemented with coated calcium-butyrate (group A); the second comprised oral vaccination with a double-attenuated Salmonella Typhimurium strain (group B), and the third was acidification of drinking water with a mixture of organic acids (group C). After challenge at 8 weeks of age, animals were individually sampled for 6 weeks (blood once per week; faeces twice per week) and then were euthanased at 14 weeks of age. Post-mortem ileum, caecum, ileocaecal lymph nodes, and tonsils were sampled, along with ileal, caecal and rectal contents, and tested for the presence of Salmonella spp. Transmission was quantified by calculating an 'adjusted' reproduction ratio 'Ra' and its 95% confidence interval (CI). The proportion of pigs that excreted Salmonella spp. via the faeces was significantly higher in group C (58%, P<0.0001) and the positive control group (41%, P=0.03), compared to group B (15%), and the proportion in group C was also significantly higher than in group A (23%, P=0.01). Group A had the lowest proportion of positive post-mortem samples (18%), followed by group B (31%), the positive control group (41%) and group C (64%) (P<0.03). The highest transmission was seen in the positive control group and group C (Ra=+∞ with 95% CI [1.88; +∞]), followed by group B (Ra=2.61 [1.21; 9.45]) and A (Ra=1.76 [1.02; 9.01]). The results of this study suggest that vaccination and supplementation of the feed with coated calcium-butyrate limited Salmonella transmission in pigs and might be useful control measures.

The nasal vestibulum is the optimal sampling site for MRSA screening in hospitalised horses.

The increased incidence of methicillin-resistant Staphylococcus aureus (MRSA) infection in equine hospitals highlights the need for infection control protocols based on optimal patient screening. In horses, the deep ventral meatus of the nasal cavity is the principal site sampled to detect MRSA. However, in humans, the anterior nares are the preferred sampling site. The objective of this study was to determine the optimal sampling location in the nasal chambers for MRSA in horses by comparing the results obtained from three different locations (the vestibulum, diverticulum and ventral meatus) in 240 hospitalised animals. Antimicrobial susceptibility testing and epidemiological typing were conducted on representative subsets of the isolates obtained. Compared to the more invasive ventral meatus sampling (relative sensitivity 68.9%; isolation rate 37.9%), vestibulum (RS 81.1%; IR 44.6%, P=0.13) and diverticulum (RS 52.3%; IR 28.8%, P=0.03) sampling were more or less sensitive, respectively. In total, 132 horses (55%) were MRSA positive with the vast majority (98.5%) carrying genotyped isolates of the livestock-associated (LA)-MRSA clonal complex (CC) 398, and only a minority (1.5%) CC8. Of the 22 MLST typed isolates, five belonged to a novel ST2197 (t011, CC398). Although 93.9% of the isolates were multi-resistant (to ß-lactam, tetracycline, trimethoprim, and gentamicin), <5% were resistant to virtually all antimicrobials commonly used in equine medicine. The study findings indicate that detection of MRSA in horses may be enhanced by replacing the traditional deep sampling of the ventral nasal meatus by the less invasive approach of sampling the nasal vestibulum.

Methicillin-resistant Staphylococcus aureus (MRSA) ST398 in pig farms and multispecies farms.

During the last few years, methicillin-resistant Staphylococcus aureus (MRSA) ST398 has been isolated frequently from livestock, especially from pigs and to a lesser extent from cattle and poultry. To gain insight into the distribution of this bacterium in pig farms versus multispecies farms, 30 Belgian farms (10 pig, 10 pig/poultry and 10 pig/cattle farms) were screened for the presence of MRSA. On each farm, 10 nasal swabs were taken from pigs. When present, cattle (n=10) were sampled in the nares and poultry (n=10) in the nares, earlobes and cloaca. A selection of the obtained isolates were further characterized using multilocus sequence typing (MLST), spa typing, SCCmec typing, pulsed field gel electrophoresis (PFGE), multiple-locus variable-number tandem repeat analysis (MLVA) and antimicrobial susceptibility testing. On 26 of 30 farms, MRSA was isolated from pigs. Furthermore, MRSA was also isolated from poultry and cattle on one pig/poultry and five pig/cattle farms, respectively. All tested MRSA isolates belonged to ST398. Eight spa types (t011, t034, t567, t571, t1451, t2974, t3423 and t5943) were detected, among which t011 predominated. SCCmec cassettes type IVa and V were present in 20% and 72% of the isolates, respectively. When combining the results of the two remaining typing methods, PFGE and MLVA, eighteen genotypes were obtained of which one genotype predominated (56% of the positive farms). All MRSA isolates were resistant to tetracycline. Resistance to trimethoprim, aminoglycosides, macrolides, lincosamides, fluoroquinolones and chloramphenicol was also observed. In conclusion, there was no effect of the farm type on the MRSA status of the pigs. A statistically significant difference was observed when comparing the pig/poultry or the pig/cattle MRSA status on the multispecies farms. Additionally, a wide variety of MRSA ST398 strains was found within certain farms when combining different typing methods.

Public health impact and antimicrobial selection of meticillin-resistant staphylococci in animals.

Whilst meticillin-resistant Staphylococcus aureus (MRSA) infections reported sporadically in companion animals and cattle in the 1970s were probably of human origin, the recently emerged livestock-associated MRSA (LA-MRSA) and meticillin-resistant Staphylococcus pseudintermedius (MRSP) strains clearly have animal origins and their isolation from humans is usually associated with exposure to livestock and companion animals, respectively. LA-MRSA is primarily an occupational health risk to farm workers and veterinarians. The risk that this zoonotic agent may spread in the community is particularly acute in countries with high livestock production and low MRSA prevalence in the human population. MRSP is primarily a threat to animal health, and cases of human infection are rare but may be overlooked in diagnostic laboratories. There is no conclusive evidence of antimicrobial selection associated with the sudden emergence of LA-MRSA and MRSP. However, the rapid global spread of these bacteria has presumably been favoured by antimicrobial selective pressure. Tetracyclines, zinc and extended-spectrum cephalosporins (including extra-label use) are the most likely selective drivers implicated in the spread of LA-MRSA, whilst increased use of broad-spectrum ß-lactams and fluoroquinolones, partly enhanced by extra-label use and the introduction of cheap generics, may have played an important role in the rapid dissemination of MRSP. Control of LA-MRSA and MRSP requires a dual approach aimed at reducing antimicrobial consumption and preventing transmission between animals and from animals to humans or vice versa. Restricted use of fluoroquinolones and cephalosporins in livestock, and national practice guidelines for rational antimicrobial use both in food and companion animals are warranted.

Assessment of human exposure to 3rd generation cephalosporin resistant E. coli (CREC) through consumption of broiler meat in Belgium.

Acquired resistance of Escherichia coli to 3rd generation cephalosporin antimicrobials is a relevant issue in intensive broiler farming. In Belgium, about 35% of the E. coli strains isolated from live broilers are resistant to 3rd generation cephalosporins while over 60% of the broilers are found to be carrier of these 3rd generation cephalosporin resistant E. coli (CREC) after selective isolation. A model aimed at estimating the exposure of the consumer to CREC by consumption of broiler meat was elaborated. This model consists of different modules that simulate the farm to fork chain starting from primary production, over slaughter, processing and distribution to storage, preparation and consumption of broiler meat. Input data were obtained from the Belgian Food Safety agencies' annual monitoring plan and results from dedicated research programs or surveys. The outcome of the model using the available baseline data estimates that the probability of exposure to 1000 colony forming units (cfu) of CREC or more during consumption of a meal containing chicken meat is ca. 1.5%, the majority of exposure being caused by cross contamination in the kitchen. The proportion of CREC (within the total number of E. coli) at primary production and the overall contamination of broiler carcasses or broiler parts with E. coli are dominant factors in the consumer exposure to CREC. The risk of this exposure for human health cannot be estimated at this stage given a lack of understanding of the factors influencing the transfer of cephalosporin antimicrobial resistance genes from these E. coli to the human intestinal bacteria and data on the further consequences of the presence of CREC on human health.

Methicillin-resistant Staphylococcus aureus (MRSA) on the skin of long-term hospitalised horses.

Given the significance of methicillin-resistant Staphylococcus aureus (MRSA) infections for both horses and staff in equine veterinary hospitals, protocols are required to minimise the risk of nosocomial transmission, including the screening of the skin and nasal chambers of equine patients for evidence of infection. The objective of this study was to clarify the potential existence and extent of MRSA on the skin of horses requiring long-term hospitalisation (≥ 6 months). Thirty such horses were sampled at eight different locations on their skin and from their nasal chambers. MRSA was isolated from 12 animals (40%), with all sample sites testing positive on at least one occasion. Organisms were most frequently detected in the nasal chambers (relative sensitivity, 83.3%; 34.5% positive horses; isolation rate 33.3%). Skin presence was found in 30% of animals with the highest isolation rates found at the carpus (16.7%), neck, withers and croup (13.3% each). To achieve a relative screening sensitivity of >90%, at least one skin site was required in addition to nasal sampling. This evidence of skin as well as nasal reservoirs of MRSA in long-term hospitalised horses should facilitate the design of effective screening and containment protocols.

Risk factors for ceftiofur resistance in Escherichia coli from Belgian broilers.

A cross-sectional study on 32 different Belgian broiler farms was performed in 2007 and 2008 to identify risk factors for ceftiofur resistance in Escherichia coli. On each farm, one E. coli colony was isolated from 30 random birds. Following susceptibility testing of 14 antimicrobials, an on-farm questionnaire was used to obtain information on risk factors. Using a multilevel logistic regression model two factors were identified at the animal level: resistance to amoxicillin and to trimethoprim-sulfonamide. On the farm level, besides antimicrobial use, seven management factors were found to be associated with the occurrence of ceftiofur resistance in E. coli from broilers: poor hygienic condition of the medicinal treatment reservoir, no acidification of drinking water, more than three feed changes during the production cycle, hatchery of origin, breed, litter material used, and treatment with amoxicillin. This study confirms that not only on-farm antimicrobial therapy, but also management- and hatchery-related factors influence the occurrence of antimicrobial resistance.

Diversity of accessory genome of human and livestock-associated ST398 methicillin resistant Staphylococcus aureus strains.

BACKGROUND: Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) has documented the diversity of the genetic background of strains associated with healthcare (HA-MRSA), community (CA-MRSA) and livestock (LA-MRSA). The accessory and core-variable genomes of those strains however remain largely unknown. OBJECTIVE: To compare the genetic background and accessory and core variable gene content of ST398 LA-MRSA strains with those of HA-and CA-MRSA strains from the same region. METHODS: Representative strains of HA- (n=21), CA- (n=13) and ST398 LA-MRSA (n=18) were selected from Belgian National Reference Laboratory collections. The accessory and core-variable genomes of these strains were characterized by a DNA-microarray composed of oligonucleotide probes targeting ~400 resistance, adhesion and virulence associated genes. RESULTS: ST398 strains displayed very homogenous hybridization profiles irrespective of their host origin. This ST398 genomic profile was moderately related to that of certain human HA- or CA-lineages but distinctively lacked several virulence- and colonization-associated genes implicated in carriage in humans, such as proteases and adhesins. No enterotoxin gene was found among ST398 strains. Differences were observed in the mobile resistance gene content of ST398 strains, including antibiotic resistance determinants. CONCLUSION: LA-MRSA strains represent a homogenous lineage distinct from co-local HA- and CA-MRSA strains, especially in its accessory genome content characterized by a lack of human-associated virulence and adhesion determinants. The absence of detectable enterotoxin gene among ST398 LA-MRSA strains from a wide host range is reassuring regarding their foodborne pathogenic potential.

In situ ESBL conjugation from avian to human Escherichia coli during cefotaxime administration.

AIMS: The behaviour of an Escherichia coli isolate of broiler origin harbouring a bla(TEM-52) -carrying plasmid (lactose-negative mutant of B1-54, IncII group) was studied in an in situ continuous flow culture system, simulating the human caecum and the ascending colon during cefotaxime administration. METHODS AND RESULTS: Fresh faeces from a healthy volunteer, negative for cephalosporin-resistant E. coli, were selected to prepare inocula. The microbiota was monitored by plating on diverse selective media, and a shift in the populations of bacteria was examined by 16S rDNA PCR denaturing gradient gel electrophoresis. Escherichia coli transconjugants were verified by plasmid and pulsed-field gel electrophoresis profiles (PFGE). The avian extended-spectrum ß-lactamase-positive E. coli was able to proliferate without selective pressure of cefotaxime, and E. coli transconjugants of human origin were detected 24 h after inoculation of the donor strain. Upon administration of cefotaxime to the fresh medium, an increase in the population size of E. coli B1-54 and the transconjugants was observed. PFGE and plasmid analysis revealed a limited number of human E. coli clones receptive for the bla(TEM-52) -carrying plasmid. CONCLUSIONS: These observations provide evidence of the maintenance of an E. coli strain of poultry origin and the horizontal gene transfer in the human commensal bowel microbiota even without antimicrobial treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The fact that an E. coli strain of poultry origin might establish itself and transfer its bla gene to commensal human E. coli raises public health concerns.

Methicillin-resistant Staphylococcus aureus (MRSA) in food production animals.

Until recently, reports on methicillin-resistant Staphylococcus aureus (MRSA) in food production animals were mainly limited to occasional detections in dairy cattle mastitis. However, since 2005 a MRSA clone, CC398, has been reported colonizing pigs, veal calves and broiler chickens and infecting dairy cows. Many aspects of its prevalence in pigs remain unclear. In other livestock, colonizing capacity and reservoir status still require elucidation. MRSA CC398 has also been detected in meat, but, as for other MRSA, the risk this poses is somewhat unclear. Currently, the most worrying aspect of MRSA CC398 appears to be its capacity to spread to humans. This might complicate MRSA control measures in human healthcare, urging research into risk factors and transmission routes. Although infections with MRSA CC398 are much less reported than carriage, more investigation into its pathogenic potential is required. Moreover, the origin and evolution of this clone remain unknown.