Metagenomics of antimicrobial and heavy metal resistance in the cecal microbiome of fattening pigs raised without antibiotics.

This study aimed to detect the cecal microbiome, antimicrobial resistance (AMR) and heavy metal resistance genes (MRGs) in fattening pigs raised under antibiotic-free (ABF) conditions compared with ordinary industrial pigs (control, C) using whole-genome shotgun sequencing. ABF pigs showed the enrichment of Prevotella (33%) and Lactobacillus (13%), whereas Escherichia coli (40%), Fusobacterium and Bacteroides (each at 4%) were notably observed in the C group. Distinct clusters of cecal microbiota of ABF and C pigs were revealed; however, microbiota of some C pigs (C1) appeared in the same cluster as ABF and were totally separated from the remaining C pigs (C2). For AMR genes, the highest abundance tet(Q) (35.7%) and mef(A) (12.7%) were markedly observed in the ABF group whereas tet(Q) (26.2%) and tet(W) (10.4%) were shown in the C group. tet(Q) was positively correlated to Prevotella in ABF and C1 samples. In the C2 group, the prominent tet(W) was positively correlated to Fusobacterium and Bacteroides Pigs have never received tetracycline but pregnant sows used chlortetracycline once 7 d before parturition. Chromosomal Cu and Zn resistance genes were also shown in both groups regardless the received Cu and Zn feed additives. A higher abundance of multi-metal resistance genes was observed in the C group (44%) compared with the ABF group (41%). In conclusion, the microbiome clusters in some C pigs were similar to that in ABF pigs. High abundant tetracycline resistance genes interrelated to major bacteria were observed in both ABF and C pigs. MRGs were also observed.IMPORTANCE: Owing to the increased problem of AMR in farm animals, raising farm animals without antibiotics is one method that could solve this problem. Our study showed that only some tetracycline and macrolide resistance genes, tet(Q), tet(W) and mef(A), were markedly abundant in ABF and C groups. The tet(Q) and tet(W) genes interrelated to different predominant bacteria in each group, showing the potential role of major bacteria as reservoirs of AMR genes. In addition, chromosomal Cu and Zn resistance genes were also observed in both pig groups, not depending on the use of Cu and Zn additives in both farms. The association of MRGs and AMR genotypes and phenotypes together with the method to re-sensitize bacteria to antibiotics should be studied further to unveil the cause of high resistance genes and solve the problems.

Construction, expression and purification of a novel CadF-based multiepitope antigen and its immunogenic polyclonal antibody specific to Campylobacter jejuni and Campylobacter coli.

Campylobacteriosis is a disease in humans caused by the infection from Campylobacter spp. Human cases are mainly due to Campylobacter jejuni, although C. coli can cause gastroenteritis in humans as well. The bacteria are commensal in chicken tract and can be contaminated into chicken products during processing. Obviously, detecting reagents such as a specific antibody is essential for the development of immune-based detection methods for C. jejuni or C. coli. In this study, in silico techniques were used to design a chimeric recombinant antigen, named multiepitope antigen (MEA), for the production of specific polyclonal antibody. To design MEA polypeptide based on C. jejuni fibronectin-binding protein or CadF, four conserved and unique antigenic peptides were identified and fused together directly. The C. jejuni CadF-based MEA polypeptide fused with two single six-histidine tags at both C- and N-terminal ends was expressed under Escherichia coli expression system. The recombinant MEA was successfully produced and purified by Ni-NTA resin with a high satisfactory yield. Indirect ELISA results showed that anti-MEA polyclonal antibody derived from rabbit serum had a titer of 16,000, indicating high antigenicity of MEA polypeptide. Dot blot results also confirmed that the produced anti-MEA antibody could specifically recognize both C. jejuni and C. coli whole cells as expected while there was no cross-reactivity to non-Campylobacter spp. tested in this study.

Identification of a novel membrane transporter mediating resistance to organic arsenic in Campylobacter jejuni.

Although bacterial mechanisms involved in the resistance to inorganic arsenic are well understood, the molecular basis for organic arsenic resistance has not been described. Campylobacter jejuni, a major food-borne pathogen causing gastroenteritis in humans, is highly prevalent in poultry and is reportedly resistant to the arsenic compound roxarsone (4-hydroxy-3-nitrobenzenearsonic acid), which has been used as a feed additive in the poultry industry for growth promotion. In this study, we report the identification of a novel membrane transporter (named ArsP) that contributes to organic arsenic resistance in Campylobacter. ArsP is predicted to be a membrane permease containing eight transmembrane helices, distinct from other known arsenic transporters. Analysis of multiple C. jejuni isolates from various animal species revealed that the presence of an intact arsP gene is associated with elevated resistance to roxarsone. In addition, inactivation of arsP in C. jejuni resulted in 4- and 8-fold reductions in the MICs of roxarsone and nitarsone, respectively, compared to that for the wild-type strain. Furthermore, cloning of arsP into a C. jejuni strain lacking a functional arsP gene led to 16- and 64-fold increases in the MICs of roxarsone and nitarsone, respectively. Neither mutation nor overexpression of arsP affected the MICs of inorganic arsenic, including arsenite and arsenate, in Campylobacter. Moreover, acquisition of arsP in NCTC 11168 led to accumulation of less roxarsone than the wild-type strain lacking arsP. Together, these results indicate that ArsP functions as an efflux transporter specific for extrusion of organic arsenic and contributes to the resistance to these compounds in C. jejuni.

Presence and characterization of blaNDM-1-positive carbapenemase-producing Klebsiella pneumoniae from outpatients in Thailand.

BACKGROUND: Presently, community-associated carbapenemase-producing Enterobacterales (CPE) remains largely unknown and require public attention. This study aimed to investigate the presence of CPE from outpatients in Thailand. METHODS: Non-duplicate stool (n = 886) and urine (n = 289) samples were collected from outpatients with diarrhea and urinary tract infection, respectively. Demographic data and characteristics of patients were collected. Isolation of CPE was performed by plating enrichment culture on agar supplemented with meropenem. Carbapenemase genes were screened by PCR and sequencing. CPE isolates were phenotypically and genotypically characterized. RESULTS: Fifteen samples (1.3%, 14 stool and 1 urine) yielded blaNDM-1-positive carbapenemase-producing Klebsiella pneumoniae (CPKP). Additional resistance to colistin and tigecycline was observed in 53.3% and 46.7% of isolates, respectively. Age >60 years was identified as a risk factor for patients with CPKP (P < 0.001, adjusted odds ratio = 11.500, 95% confidence interval = 3.223-41.034). Pulsed field gel electrophoresis revealed genetic diversity of CPKP isolates; however, clonal spread has been observed. ST70 (n = 4) was common, followed by ST147 (n = 3). blaNDM-1 from all isolates were transferable and mainly resided on IncA/C plasmid (80%). All blaNDM-1 plasmids remained stable in bacterial host for at least 10 days in antibiotic-free environments, regardless of replicon types. CONCLUSION: This study demonstrates that the prevalence of CPE among outpatients in Thailand remains low and the spread of blaNDM-1-positive CPKP may be driven by IncA/C plasmid. Our results emphasize the need for a large-scale surveillance study to limit further spread of CPE in community.

Fine Particle Adsorption Capacity of Volcanic Soil from Southern Kyushu, Japan.

"Akahoya" is a volcanic soil classified as a special soil deposited in Kyushu, Japan. Many of its properties are not yet clearly understood. We found that Akahoya had the potential to adsorb bacteria in cattle feces, which prompted us to investigate its material properties and perform experiments to comprehensively evaluate its adsorption performance for various fine particles such as acidic and basic dyes, NOx/SOx gas, and phosphoric acid ions, in addition to bacteria. Akahoya had a very high specific surface area owing to the large number of nanometer-sized pores in its structure; it exhibited a high adsorption capacity for both NO2 and SO2. Regarding the zeta potential of Akahoya, the point of zero charge was approximately pH 7.0. The surface potential had a significant effect on the adsorption of acidic and basic dyes. Akahoya had a very high cation exchange capacity when the sample surface was negatively charged and a high anion exchange capacity when the sample surface was positively charged. Akahoya also exhibited a relatively high adsorption capacity for phosphoric acid because of its high level of Al2O3, and the immersion liquid had a very high Al ion concentration. Finally, filtration tests were performed on Escherichia coli suspension using a column filled with Akahoya or another volcanic soil sample. The results confirmed that the Escherichia coli adhered on the Akahoya sample. The results of the Escherichia coli release test, after the filtration test, suggested that this adhesion to Akahoya could be phosphorus-mediated.

Impaired fitness and transmission of macrolide-resistant Campylobacter jejuni in its natural host.

Campylobacter jejuni is a major zoonotic pathogen transmitted to humans via the food chain and is prevalent in chickens, a natural reservoir for this pathogenic organism. Due to the importance of macrolide antibiotics in clinical therapy of human campylobacteriosis, development of macrolide resistance in Campylobacter has become a concern for public health. To facilitate the control of macrolide-resistant Campylobacter, it is necessary to understand if macrolide resistance affects the fitness and transmission of Campylobacter in its natural host. In this study we conducted pairwise competitions and comingling experiments in chickens using clonally related and isogenic C. jejuni strains, which are either susceptible or resistant to erythromycin (Ery). In every competition pair, Ery-resistant (Ery(r)) Campylobacter was consistently outcompeted by the Ery-susceptible (Ery(s)) strain. In the comingling experiments, Ery(r) Campylobacter failed to transmit to chickens precolonized by Ery(s) Campylobacter, while isogenic Ery(s) Campylobacter was able to transmit to and establish dominance in chickens precolonized by Ery(r) Campylobacter. The fitness disadvantage was linked to the resistance-conferring mutations in the 23S rRNA. These findings clearly indicate that acquisition of macrolide resistance impairs the fitness and transmission of Campylobacter in chickens, suggesting that the prevalence of macrolide-resistant C. jejuni will likely decrease in the absence of antibiotic selection pressure.

Live-Attenuated Oral Vaccines to Reduce Campylobacter Colonization in Poultry.

The control of Campylobacter in poultry at the pre-harvest level is critical to reducing foodborne infections with Campylobacter since the consumption of contaminated poultry is the most frequent cause of human campylobacteriosis. Although poultry vaccination is suggested as useful intervention measures, no Campylobacter vaccines are currently available. To develop live-attenuated oral Campylobacter vaccines, in this study, we evaluated the efficacy of pre-colonization by oxidative stress defense mutants, including knockout mutants of ahpC, katA, and sodB, in preventing Campylobacter jejuni from colonizing poultry. Interestingly, when chickens were pre-colonized with ΔahpC and ΔkatA mutants, rather than the ΔsodB mutant, the level of C. jejuni colonization was significantly reduced within 35 days. Further studies demonstrated when chickens were pre-colonized with the ΔahpC mutant by oral challenge with a high dose (ca., 5 × 108 CFU/bird) and a low dose (ca., 5 × 106 CFU/bird), it twice reduced the level of C. jejuni by 3.9 log10CFU/g feces and 3 log10CFU/g feces after 42 days, respectively, compared to the untreated control. Due to a colonization defect, the ΔahpC mutant was removed from chickens within 42 days. After excretion from the host, moreover, the ΔahpC mutant cannot survive in aerobic environments because of compromised aerotolerance. Our findings suggest that the ahpC mutant has a great potential for on-farm application to control C. jejuni at the pre-harvest level.

Research Note: Longitudinal fecal shedding patterns and characterization of Salmonella enterica and mcr-positive Escherichia coli in meat-type ducks raised in an open-house system.

This longitudinal study aimed to determine the fecal shedding pattern and characterize Salmonella enterica and mcr-positive Escherichia coli from meat-type ducks raised in an open-house system in Thailand. Fecal samples (n = 1,475) were collected from ducks over a 6-month period. Overall, the detection rate of S. enterica was 5.4% and the highest fecal shedding rate was noted in 4-day-old ducklings (28.8%). Then, S. enterica shedding decreased to 10, 8, 4.7, and 0.7% when ducks reached the ages of 10 d, 17 d, 3 wk, and 4 wk, respectively. Seventy-nine isolates were recovered and Salmonella Amsterdam was the predominant serovar (79.7%). With respect to colistin-resistant E. coli, mcr-positive E. coli (colistin MICs = 8-16 µg/mL) was noted in ducks at the ages of 16 wk (6.0%) and 24 wk (18.7%). mcr-1 was the most common (75.7%), followed by mcr-3 (13.5%), and mcr-1 and mcr-3 co-carriage (10.8%). Most S. enterica isolates were susceptible to antibiotics and multidrug resistant (MDR) was found in only a single isolate. However, as many as 89.2% of mcr-positive E. coli were defined as MDR. Almost all S. enterica isolates (97.5-100%) carried several virulence genes involving in invasion, intracellular survival, and iron metabolism. Pulsed Field Gel Electrophoresis revealed that several mcr-positive E. coli isolates were clonally unrelated. Conjugative transfer of mcr-1, mcr-3 as well as co-transfer of mcr-1 and mcr-3 was observed with the frequencies ranging from 10-8 to 10-3. All mcr-1 resided on IncI2, while mcr-3 was associated with IncF and IncX4 plasmids. Our study provides the evidence of fecal shedding pattern of S. enterica and mcr-positive E. coli from meat-type ducks, highlighting the importance of duck farming in the dissemination of pathogenic bacteria that are potentially hazardous to human.

Risk Factors for Community-Acquired Urinary Tract Infections Caused by Multidrug-Resistant Enterobacterales in Thailand.

The dissemination of multidrug-resistant Enterobacterales (MDRE) in community settings is becoming a great concern. This study aimed to assess the incidence and risk factors associated with community-acquired urinary tract infections (CA-UTIs) caused by MDRE. A prospective case−control study was undertaken among patients with UTIs visiting an outpatient department in Phitsanulok Province, Thailand. Urine samples were collected and screened to include only patients with Enterobacterales infections. Risk factors were analyzed by multivariate logistic regression analysis. Of the 284 patients with CA-UTIs, 25.7% (n = 73) and 74.3% (n = 211) were positive for MDRE (case) and non-MDRE (control), respectively. Being a farmer was identified as an independent risk factor for MDRE-associated CA-UTIs (adjusted odds ratio = 3.101; 95% confidence interval = 1.272−7.564; p = 0.013). A total of 309 Enterobacterales isolates were recovered, and Escherichia coli was the most frequently detected (86.4%). The highest resistance rate was observed for ampicillin (67.0%), followed by ciprofloxacin (34.0%) and cotrimoxazole (32.7%), while resistance to third-generation cephalosporins (cefotaxime, ceftriaxone) and levofloxacin remained <20%. Resistance to ampicillin−gentamicin−cotrimoxazole was the most common pattern among MDRE isolates. Interestingly, we detected a colistin-resistant Enterobacter cloacae harboring mcr-9 (colistin MIC = 16 µg/mL). mcr-9 was transferable at high frequency (4.5 × 10−4) and resided on IncF plasmid. This study demonstrates that being a farmer is a risk factor for MDRE-associated CA-UTIs. Interestingly, this is the first report to identify mcr-9-positive E. cloacae from a Thai patient in the community.

The worldwide trend of Campylobacter spp., infection from duck-related isolates and associated phenotypic and genotypic antibiotic resistance, since 1985: identifying opportunities and challenges for prevention and control.

Campylobacter, a leading cause of foodborne diseases, is well recognized worldwide. Poultry and poultry products are considered as major sites for Campylobacter infection in humans. The extensive uses of antibiotics mostly as growth promoters and for therapeutic purposes have led to the emergence of antibiotic-resistant strains of foodborne pathogens including Campylobacter. A key tenet of this paper is the need for reviewing the previous studies conducted around the globe on the prevalence and antimicrobial resistance of Campylobacter spp. isolates in duck to better understand the sources and trends of infection. Based on published data, the prevalence of Campylobacter spp. in duck and duck-related samples ranged from 0% to 100% and was largely influenced by the isolation method. Among Campylobacter spp., C. jejuni was the predominant cause of campylobacteriosis, followed by C. coli. Campylobacter spp. from ducks were mostly resistant to fluoroquinolones and tetracycline and a lesser extent to gentamicin, chloramphenicol, and erythromycin. Some studies showed that ducks may pose a risk for acquiring campylobacteriosis because they had genotypes quite similar to human isolates detected previously. A continued monitoring approach is needed, at national and international levels, with enhanced surveillance and reporting of trends, as well as harmonization of surveillance systems toward a one-health approach to monitoring antimicrobial resistance in animal production particularly if increased resistance rates are being demonstrated.

Rapid and simultaneous detection of Campylobacter spp. and Salmonella spp. in chicken samples by duplex loop-mediated isothermal amplification coupled with a lateral flow biosensor assay.

Development of a simple, rapid and specific assay for the simultaneous detection of Campylobacter spp. and Salmonella spp. based on duplex loop-mediated isothermal amplification (d-LAMP), combined with lateral-flow biosensor (LFB) is reported herein. LAMP amplicons of both pathogens were simultaneously amplified and specifically differentiated by LFB. The specificity of the d-LAMP-LFB was evaluated using a set of 68 target and 12 non-target strains, showing 100% inclusivity and exclusivity. The assay can simultaneously detect Campylobacter and Salmonella strains as low as 1 ng and 100 pg genomic DNA per reaction, respectively. The lowest inoculated detection limits for Campylobacter and Salmonella species in artificially contaminated chicken meat samples were 103 CFU and 1 CFU per 25 grams, respectively, after enrichment for 24 h. Furthermore, compared to culture-based methods using field chicken meat samples, the sensitivity, specificity and accuracy of d-LAMP- LFB were 95.6% (95% CI, 78.0%-99.8%), 71.4% (95% CI, 29.0%-96.3%) and 90.0% (95% CI, 73.4%-97.8%), respectively. The developed d-LAMP-LFB assay herein shows great potentials for the simultaneous detection of the Campylobacter and Salmonella spp. and poses a promising alternative approach for detection of both pathogens with applications in food products.

Prevalence of tetracycline-resistant Campylobacter in organic broilers during a production cycle.

Tetracycline (tet) resistance in Campylobacter isolated from organically raised broilers was investigated in this study. Two hundred forty-five samples from an organic broiler farm were collected weekly from the first week to the end of the production cycle, and they were cultured for thermophilic Campylobacter. Tetracycline resistance of these Campylobacter isolates was identified by the agar dilution method, whereas DNA fingerprinting profiles of tet-susceptible and tet-resistant strains were determined by pulsed-field gel electrophoresis (PFGE). None of the Campylobacter isolates from the third and the fourth week of the production period were resistant to tetracycline, whereas 66.7% of the isolates from the fifth week were resistant to this antibiotic. Although the prevalence of tetracycline resistance reached 100.0% during the sixth and seventh week, less than 34.0% of the isolates from the 10th week were resistant to this antimicrobial agent. In addition, only 13.8% of Campylobacter isolates from the intestinal tracts of these organically raised broilers were resistant to tetracycline. The presence of the tet(O) gene was detected in 98.9% of tet-resistant Campylobacter isolates, and tet-susceptible and tet-resistant Campylobacter strains showed distinct PFGE genotypes. The results suggest that the Campylobacter strains isolated from the early stage of the production were susceptible to tetracycline, but they were subsequently displaced by tet-resistant Campylobacter.

Distribution and Genetic Profiles of Campylobacter in Commercial Broiler Production from Breeder to Slaughter in Thailand.

Poultry and poultry products are commonly considered as the major vehicle of Campylobacter infection in humans worldwide. To reduce the number of human cases, the epidemiology of Campylobacter in poultry must be better understood. Therefore, the objective of the present study was to determine the distribution and genetic relatedness of Campylobacter in the Thai chicken production industry. During June to October 2012, entire broiler production processes (i.e., breeder flock, hatchery, broiler farm and slaughterhouse) of five broiler production chains were investigated chronologically. Representative isolates of C. jejuni from each production stage were characterized by flaA SVR sequencing and multilocus sequence typing (MLST). Amongst 311 selected isolates, 29 flaA SVR alleles and 17 sequence types (STs) were identified. The common clonal complexes (CCs) found in this study were CC-45, CC-353, CC-354 and CC-574. C. jejuni isolated from breeders were distantly related to those isolated from broilers and chicken carcasses, while C. jejuni isolates from the slaughterhouse environment and meat products were similar to those isolated from broiler flocks. Genotypic identification of C. jejuni in slaughterhouses indicated that broilers were the main source of Campylobacter contamination of chicken meat during processing. To effectively reduce Campylobacter in poultry meat products, control and prevention strategies should be aimed at both farm and slaughterhouse levels.

Antibiotic resistance in Campylobacter: emergence, transmission and persistence.

Campylobacter is a leading foodborne bacterial pathogen, which causes gastroenteritis in humans. This pathogenic organism is increasingly resistant to antibiotics, especially fluoroquinolones and macrolides, which are the most frequently used antimicrobials for the treatment of campylobacteriosis when clinical therapy is warranted. As a zoonotic pathogen, Campylobacter has a broad animal reservoir and infects humans via contaminated food, water or milk. Antibiotic usage in both animal agriculture and human medicine, can influence the development of antibiotic-resistant Campylobacter. This review will describe the trend in fluoroquinolone and macrolide resistance in Campylobacter, summarize the mechanisms underlying the resistance to various antibiotics and discuss the unique features associated with the emergence, transmission and persistence of antibiotic-resistant Campylobacter. Special attention will be given to recent findings and emphasis will be placed on Campylobacter resistance to fluoroquinolones and macrolides. A future perspective on antibiotic resistance and potential approaches for the control of antibiotic-resistant Campylobacter, will also be discussed.

Comparison of antimicrobial susceptibility testing of Campylobacter spp. by the agar dilution and the agar disk diffusion methods.

The correlation and the level of agreement between the standardized agar dilution and the agar disk diffusion methods for antimicrobial susceptibility testing of Campylobacter were investigated. A high-level agreement between the two methods was evident for aminoglycosides and fluoroquinolones, while a low-level agreement was observed for other antibiotics.

Effect of conventional and organic production practices on the prevalence and antimicrobial resistance of Campylobacter spp. in poultry.

Intestinal tracts of broilers and turkeys from 10 conventional broiler farms and 10 conventional turkey farms, where antimicrobials were routinely used, and from 5 organic broiler farms and 5 organic turkey farms, where antimicrobials had never been used, were collected and cultured for Campylobacter species. A total of 694 Campylobacter isolates from the conventional and organic poultry operations were tested for antimicrobial resistance to nine antimicrobial agents by the agar dilution method. Although Campylobacter species were highly prevalent in both the conventional and organic poultry operations, the antimicrobial resistance rates were significantly different between the organic operations and the conventional operations. Less than 2% of Campylobacter strains isolated from organically raised poultry were resistant to fluoroquinolones, while 46% and 67% of Campylobacter isolates from conventionally raised broilers and conventionally raised turkeys, respectively, were resistant to these antimicrobials. In addition, a high frequency of resistance to erythromycin (80%), clindamycin (64%), kanamycin (76%), and ampicillin (31%) was observed among Campylobacter isolates from conventionally raised turkeys. None of the Campylobacter isolates obtained in this study was resistant to gentamicin, while a large number of the isolates from both conventional and organic poultry operations were resistant to tetracycline. Multidrug resistance was observed mainly among Campylobacter strains isolated from the conventional turkey operation (81%). Findings from this study clearly indicate the influence of conventional and organic poultry production practices on antimicrobial resistance of Campylobacter on poultry farms.

Molecular typing of Campylobacter strains using the cmp gene encoding the major outer membrane protein.

Thermophilic Campylobacter, particularly Campylobacter jejuni, is one of the major foodborne human pathogens of animal origin. Reliable and sensitive typing tools are required for understanding the epidemiology and ecology of this zoonotic bacteria agent. Currently, several molecular typing methods are available for differentiating Campylobacter strains, but each of them has limitations. Our previous study revealed that considerable sequence polymorphism exists in the cmp gene encoding the major outer membrane protein of Campylobacter and suggested that sequence variation of cmp may be utilized for discrimination of Campylobacter strains. In this study, we evaluated the feasibility of the cmp-based typing tool, using pulsed-field gel electrophoresis (PFGE) as the "gold" standard for comparison. The cmp alleles were sequenced from multiple Campylobacter strains, grouped, and compared with the PFGE profiles of these strains using Bionumerics. Results showed that 43 cmp sequence types and 43 PFGE types existed among the 60 Campylobacter isolates. Typeability of these strains is 100% using either the cmp-based method or PFGE. The discrimination indices are 0.973 for the cmp-based method and 0.969 for PFGE, respectively. The cmp sequence types are 77.6% congruent with the PFGE types. These results indicate that the cmp-based typing is a simple, yet highly discriminatory approach for molecular differentiation of C. jejuni strains.