Characterization Of Antimicrobial Resistance Genes Research Articles

Core and variable antimicrobial resistance genes in the gut microbiomes of Chinese and European pigs.

Monitoring the prevalence of antimicrobial resistance genes (ARGs) is vital for addressing the global crisis of antibiotic-resistant bacterial infections. Despite its importance, the characterization of ARGs and microbiome structures, as well as the identification of indicators for routine ARG monitoring in pig farms, are still lacking, particularly concerning variations in antimicrobial exposure in different countries or regions. Here, metagenomics and random forest machine learning were used to elucidate the ARG profiles, microbiome structures, and ARG contamination indicators in pig manure under different antimicrobial pressures between China and Europe. Results showed that Chinese pigs exposed to high-level antimicrobials exhibited higher total and plasmid-mediated ARG abundances compared to those in European pigs ( P<0.05). ANT(6)-Ib, APH(3')-IIIa, and tet(40) were identified as shared core ARGs between the two pig populations. Furthermore, the core ARGs identified in pig populations were correlated with those found in human populations within the same geographical regions. Lactobacillus and Prevotella were identified as the dominant genera in the core microbiomes of Chinese and European pigs, respectively. Forty ARG markers and 43 biomarkers were able to differentiate between the Chinese and European pig manure samples with accuracies of 100% and 98.7%, respectively. Indicators for assessing ARG contamination in Chinese and European pigs also achieved high accuracy ( r=0.72-0.88). Escherichia flexneri in both Chinese and European pig populations carried between 21 and 37 ARGs. The results of this study emphasize the importance of global collaboration in reducing antimicrobial resistance risk and provide validated indicators for evaluating the risk of ARG contamination in pig farms.

Molecular characterization of antimicrobial resistance genes of Staphylococcus aureus isolated from mastitic camel milk in Egypt.

Staphylococcus aureus is one of the most common causes of mastitis worldwide. This study aimed to determine the prevalence and antimicrobial resistance (AMR) patterns of S. aureus in mastitic milk samples collected from camel farms in Matrouh Governorate, Egypt. A total of 200 mastitic camel milk samples were evaluated for S. aureus using both conventional culture-based and molecular-based methods. Antibiotic susceptibility testing of S. aureus isolates was conducted using disc diffusion and agar dilution methods, with antibiotic resistance genes identified through polymerase chain reaction with specific primers. Out of samples tested, 60 (30.00%) were positive for S. aureus. The isolates displayed the highest of resistance against piperacillin-tazobactam (55.00%) followed by trimethoprim- sulfamethoxazole (45.00%) and amoxicillin (40.00%). Half of the isolates were multidrug-resistant (MDR). The AMR genes included methicillin-resistant gene (mecA), β-lactamase gene (blaZ), tetracycline resistance gene (tetK), erythromycin resistance gene (ermB) and vancomycin resistant gene (vanA) were detected in 100%, 100%, 95.00%, 90.00% and 20.00% of the isolates, respectively. In conclusion, the presence of MDRS aureus as a cause of clinical camel mastitis is a significant veterinary and public health concern. These findings highlight the importance of proper antibiotic use in Egyptian camel farms and the need for molecular techniques to fully understand the genetic profile of antimicrobial-resistant S. aureus isolates.

Characterization of Antimicrobial Resistance Genes and Virulence Factors in the Genomes of Escherichia coli ST69 Isolates from Preweaned Dairy Calves and Their Phylogenetic Relationship with Poultry and Human Clinical Strains.

Escherichia coli sequence type 69 (ST69) are common causative agents of extraintestinal infections occurring in the bloodstream, cerebrospinal fluid, surgical sites, and, most frequently, the urinary tract. The objective of this study was to analyze the genomic characteristics of 45 antimicrobial-resistant Escherichia coli ST69 strains that were isolated from 28 calves on eight dairy farms in Pennsylvania, USA. The genomes were sequenced and the antimicrobial resistance genes (ARGs), virulence factors (VFs), and plasmid replicons were identified in silico. A phylogenetic analysis was conducted to compare these calf isolate genomes with poultry and human clinical E. coli ST69 genomes. In total, 23 ARGs, 45 VFs, and 15 plasmid replicons were identified. The majority of genomes (n = 36, 80%) had a multidrug-resistant (MDR) genotype and carried genes conferring resistance to antibiotics of human health significance. Phylogenetic analysis based on the core genomes revealed that calf isolates were nested within clades that included human and poultry isolates, indicating that they are not phylogenetically distinct. Results suggest that dairy calves are a reservoir of MDR E. coli ST69 strains with diverse ARG and VF profiles. This information will be helpful in assessing public health risks associated with E. coli ST69 in commercial dairy production systems.

Analysis of Antimicrobial Resistance Genes (ARGs) in Enterobacterales and A. baumannii Clinical Strains Colonizing a Single Italian Patient.

The dramatic increase in infections caused by critically multidrug-resistant bacteria is a global health concern. In this study, we characterized the antimicrobial resistance genes (ARGs) of K. pneumoniae, P. mirabilis, E. cloacae and A. baumannii isolated from both surgical wound and rectal swab of a single Italian patient. Bacterial identification was performed by MALDI-TOF MS and the antimicrobial susceptibility was carried out by Vitek 2 system. The characterization of ARGs was performed using next-generation sequencing (NGS) methodology (MiSeq Illumina apparatus). K. pneumoniae, P. mirabilis and E. cloacae were resistant to most β-lactams and β-lactam/β-lactamases inhibitor combinations. A. baumannii strain was susceptible only to colistin. The presence of plasmids (IncN, IncR, IncFIB, ColRNAI and Col (MGD2)) was detected in all Enterobacterales but not in A. baumannii strain. The IncN plasmid and blaNDM-1 gene were found in K. pneumoniae, P. mirabilis and E. cloacae, suggesting a possible transfer of this gene among the three clinical species. Conjugation experiments were performed using K. pneumoniae (1 isolate), P. mirabilis (2 isolates) and E. cloacae (2 isolates) as donors and E. coli J53 as a recipient. The blaNDM-1 gene was identified by PCR analysis in all transconjugants obtained. The presence of four different bacterial species harboring resistance genes to different classes of antibiotics in a single patient substantially reduced the therapeutic options.

Characterization of Antimicrobial Resistance Genes of Pasteurella multocida Isolated from Diseased Chickens in Egypt

In this study, the prevalence of Pasteurella multocida in diseased chickens, capsular genotyping, antimicrobial resistance patterns and some resistance genes were determined. Lungs, liver and spleen samples were collected from 250 diseased chickens from layers and broiler flocks from El-Gharbia and Kafr El-Sheikh governorates in Egypt for isolation of P. multocida in the period from June 2018 to December 2019. Confirmatory identification was done by using PCR for capsular type A antigen. P. multocida was isolated from 3.6 % of the diseased chicken. Six isolates of P. multocida that examined for detection of capsular type A showed positive results. Antimicrobial resistance patterns were evaluated for all isolates against twenty antimicrobial agents and the results showed 100% resistance totrimethoprim/sulfamethazole, oxacillin and nitrofurantion. Also, strains expressed highly resistant to penicillin, chloramphenicol, rifampicin and ampicillin \sulbactam, while they were sensitive to norfloxacin, clindamycin, cephalexin and cefotaxime. The antimicrobial resistance genes were detected by using PCR and the results showed that all isolates harbored β-lactam-resistantgene BlaROB-1 (100%), followed by sulfonamide resistant gene sul1 (50%), tetracycline-resistant gene tetH (33.3%) and trimethoprim-resistant dihydrofolate reductase dfrA (16.6%).

Characterization of antimicrobial resistance genes and virulence factor genes in an Arctic permafrost region revealed by metagenomics

Antimicrobial resistance genes (ARGs) and virulence factor genes (VFGs) constitute a serious threat to public health, and climate change has been predicted to affect the increase in bacterial pathogens harboring ARGs and VFGs. However, studies on bacterial pathogens and their ARGs and VFGs in permafrost region have received limited attention. In this study, a metagenomic approach was applied to a comprehensive survey to detect potential ARGs, VFGs, and pathogenic antibiotic resistant bacteria (PARB) carrying both ARGs and VFGs in the active layer and permafrost. Overall, 70 unique ARGs against 18 antimicrobial drug classes and 599 VFGs classified as 38 virulence factors were detected in the Arctic permafrost region. Eight genes with mobile genetic elements (MGEs) carrying ARGs were identified; most MGEs were classified as phages. In the metagenome-assembled genomes, the presence of 15 PARB was confirmed. The soil profile showed that the transcripts per million (TPM) values of ARGs and VFGs in the sub-soil horizon were significantly lower than those in the top soil horizon. Based on the TPM value of each gene, major ARGs, VFGs, and these genes in PARB from the Arctic permafrost region were identified and their distribution was confirmed. The major host bacteria for ARGs and VFGs and PARB were identified. A comparison of the percentage identity distribution of ARGs and VFGs to reference databases indicated that ARGs and VFGs in the Arctic soils differ from previously identified genes. Our results may help understand the characteristics and distribution of ARGs, VFGs, and these genes in PARB in the Arctic permafrost region. This findings suggest that the Arctic permafrost region may serve as potential reservoirs for ARGs, VFGs, and PARB. These genes could pose a new threat to human health if they are released by permafrost thawing owing to global warming and propagate to other regions.

Molecular characterization of antimicrobial resistance genes on farms and in commercial milk with emphasis on the effect of currently practiced heat treatments on viable but nonculturable formation

Despite the considerable advances that have been made to improve dairy food safety, there is rising concern that pasteurization is not sufficient for the destruction of plasmid-mediated antimicrobial resistance (AMR) genes of resistant bacteria and could stimulate bacteria to enter into a viable but nonculturable (VBNC) state. In the current study, we surveyed the prevalence of 1 genomic and 9 plasmid-mediated AMR genes in 100 samples (bulk tank milk and milk filter socks) at the farm level and 152 commercial milk samples (pasteurized and UHT milks) and assessed the VBNC state in dairy bacteria. Results revealed that sul2 was the most prevalent plasmid-mediated gene in milk filter socks (96%), bulk tank milk (48%), pasteurized milk (68%), and UHT (43%) milk; in contrast, mecA was not detected in any sample. Additionally, commercial pasteurization (as currently practiced) failed to decrease the prevalence of the blaTEM-B1 (43%), tetK (30%), and tetA (55%) plasmid-mediated AMR genes; thus, commercial pasteurization may be one of the factors creating the VBNC state in some dairy bacteria. Continued research is necessary to identify bacterial species entering the VBNC state after pasteurization, to assess their potential hazard level and shed more light on the expression and possibility of horizontal gene transfer of those plasmid-mediated AMR genes.

Comparative metagenomics and characterization of antimicrobial resistance genes in pasteurized and homemade fermented Arabian laban

The aim of this study was to investigate bacterial diversity and function in a fermented milk drink called laban, which is traditionally served in the Middle East, Africa, and Indian subcontinent. Pasteurized laban (LBP) and unpasteurized, homemade, raw laban (LBR) underwent 16S rRNA gene amplicon and shotgun sequencing to analyze their bacterial community, presence of antimicrobial resistance genes (ARGs), and metabolic pathways. This study highlighted relatively greater diversity in LBR bacterial populations compared to LBP, despite containing similar major taxa that consisted primarily of Firmicutes followed by Proteobacteria, Bacteroidetes, and Actinobacteria. The dominant species, Streptococcus thermophilus, was relatively more abundant in LBP (80.7%) compared to LBR (47.9%). LBR had increased diversity and higher relative abundance of several known probiotic bacteria, such as Streptococcus salivarius and Lactococcus lactis, whereas Lactobacillus acidophilus was detected at a higher abundance in LBP. Pathogens like Acinetobacter baumannii, Streptococcus pneumoniae, Streptococcus pyogenes, and Escherichia coli had lower abundance in LBP compared to LBR. Thirty-three ARGs were detected in LBR compared to nine in LBP and are responsible for resistance to 11 classes of antibiotics. A significant proportion of the metagenomes from both types of laban were assigned to housekeeping functions, such as amino acid metabolism, translation, membrane transport, and carbohydrate metabolism. LBR demonstrated increased diversity in probiotics and metabolic functions compared to LBP. However, the relatively high diversity of pathogenic and opportunistic bacteria and ARGs in LBR raises safety concerns and highlights the need for a more hygienic environment for the processing of homemade fermented dairy foods.

Characterization of antimicrobial resistance genes in Enterobacteriaceae carried by suburban mesocarnivores and locally owned and stray dogs.

The role of wildlife in the dissemination of antimicrobial-resistant bacteria and antimicrobial resistance genes (ARGs) in the environment is of increasing concern. We investigated the occurrence, richness and transmissibility potential of ARGs detected in the faeces of three mesocarnivore species: the coyote (Canis latrans), raccoon (Procyon lotor) and Virginia opossum (Didelphis virginiana), and of stray and owned dogs in suburban Chicago, IL, USA. Rectal swabs were collected from live-captured coyotes (n=32), raccoons (n=31) and Virginia opossums (n=22). Fresh faecal samples were collected from locally owned (n=13) and stray dogs (n=18) and from the live-captured mesocarnivores, when available. Faecal samples and rectal swabs were enriched to select for Enterobacteriaceae and pooled by mesocarnivore species and dog type (owned or stray). Pooled enriched samples were then analysed for the presence of ARGs using shotgun sequencing. The three mesocarnivore and stray dog samples had twice as many unique ARGs compared to the owned dog sample, which was partly driven by a greater richness of beta-lactamase genes (genes conferring resistance to penicillins and cephalosporins). Raccoon and stray dog samples had the most ARGs in common, suggesting possible exposure to similar environmental sources of ARGs. In addition to identifying clinically relevant ARGs (e.g. blaCMY and qnrB), some ARGs were linked to the class 1 integrase gene, intI1, which may indicate anthropogenic origin. Findings from this pilot investigation suggest that the microbial communities of suburban mesocarnivores and stray dogs can host ARGs that can confer resistance to several antimicrobials used in human and veterinary medicine.

Novel clinically relevant antibiotic resistance genes associated with sewage sludge and industrial waste streams revealed by functional metagenomic screening

A growing body of evidence indicates that anthropogenic activities can result in increased prevalence of antimicrobial resistance genes (ARGs) in bacteria in natural environments. Many environmental studies have used next-generation sequencing methods to sequence the metagenome. However, this approach is limited as it does not identify divergent uncharacterized genes or demonstrate activity. Characterization of ARGs in environmental metagenomes is important for understanding the evolution and dissemination of resistance, as there are several examples of clinically important resistance genes originating in environmental species. The current study employed a functional metagenomic approach to detect genes encoding resistance to extended spectrum β-lactams (ESBLs) and carbapenems in sewage sludge, sludge amended soil, quaternary ammonium compound (QAC) impacted reed bed sediment and less impacted long term curated grassland soil. ESBL and carbapenemase genes were detected in sewage sludge, sludge amended soils and QAC impacted soil with varying degrees of homology to clinically important β-lactamase genes. The flanking regions were sequenced to identify potential host background and genetic context. Novel β-lactamase genes were found in Gram negative bacteria, with one gene adjacent to an insertion sequence ISPme1, suggesting a recent mobilization event and/ the potential for future transfer. Sewage sludge and quaternary ammonium compound (QAC) rich industrial effluent appear to disseminate and/or select for ESBL genes which were not detected in long term curated grassland soils. This work confirms the natural environment as a reservoir of novel and mobilizable resistance genes, which may pose a threat to human and animal health.

Hierarchical Hidden Markov models enable accurate and diverse detection of antimicrobial resistance sequences

The characterization of antimicrobial resistance genes from high-throughput sequencing data has become foundational in public health research and regulation. This requires mapping sequence reads to databases of known antimicrobial resistance genes to determine the genes present in the sample. Mapping sequence reads to known genes is traditionally accomplished using alignment. Alignment methods have high specificity but are limited in their ability to detect sequences that are divergent from the reference database, which can result in a substantial false negative rate. We address this shortcoming through the creation of Meta-MARC, which enables detection of diverse resistance sequences using hierarchical, DNA-based Hidden Markov Models. We first describe Meta-MARC and then demonstrate its efficacy on simulated and functional metagenomic datasets. Meta-MARC has higher sensitivity relative to competing methods. This sensitivity allows for detection of sequences that are divergent from known antimicrobial resistance genes. This functionality is imperative to expanding existing antimicrobial gene databases.

Characterization of antimicrobial resistance genes and class 1 integrase gene in raw meat and aquatic product, fresh vegetable and fruit, and swine manure in southern China

Various antimicrobial resistance (AMR) genes, especially a widely diverse assembly of mobile genetic elements such as plasmids, integrons, gene cassettes, or transposons, recently identified environmental pollutants, have been widely recognized as one of the main global health concerns, threatening food safety, and human and animal health. In order to determine the contamination of AMR genes and class 1 integrase gene (intI1) in raw or fresh food and swine manure in southern China, 119 samples of raw or fresh foods were investigated (raw meat and aquatic product, fresh vegetables and fruits) and 10 samples of swine manures from Guangzhou and Xiamen were analyzed for the prevalence and distribution of 11 AMR genes and intI1 using real-time PCR. The 11 AMR genes belonged to tetracycline (tetA, tetB, tetM), aminoglycoside (aadA, aphA-1), sulfonamide (sulI, sulII), chloromycetin (cmlA, floR), erythromycin (ermB), and disinfectant resistance genes (qacE). TetA was the most frequently detected gene in food samples, with a frequency of 100% in all samples. The detection frequencies of aadA, aph-1, floR, sulI, intI1, qacE, sulII, tetB, and tetM in food samples were above 90%, whereas cmlA and ermB had lower detection frequencies (89.9% and 84.0%, respectively). Tetracycline and aminoglycoside resistance genes were the most prevalent genes in food samples, with an average abundance of 3.08 and 1.18 copies/16S rRNA gene copy, respectively. Principal component analysis revealed that the comprehensive contamination of AMR genes in aquatic products was different from that of agricultural products. IntI1 of class 1 integron, and its most commonly contain resistance cassettes encoding aadA genes, as well as cmlA, qacE, sulI, and sulII genes, were present in significantly lower abundance in aquatic than agricultural products. Dominant AMR genes in swine manure samples were tetM, qacE, aadA, and cmlA, with an average abundance above 107copies/g. The high prevalence of AMR genes and intI1 in raw or fresh food highlighted the roles of raw or fresh foods as potential sources for antimicrobial resistance genes and raised the need for controlling AMR genes transmission through the food chain.

Emergence of Resistance to Quinolones and β-Lactam Antibiotics in Enteroaggregative and Enterotoxigenic Escherichia coli Causing Traveler's Diarrhea.

The objective of this study was to assess the antimicrobial resistance of enteroaggregative Escherichia coli (EAEC) and enterotoxigenic E. coli (ETEC) strains causing traveler's diarrhea (TD) and to investigate the molecular characterization of antimicrobial resistance genes to third-generation cephalosporins, cephamycins, and quinolones. Overall, 39 EAEC and 43 ETEC clinical isolates were studied. The susceptibilities of EAEC and ETEC against ampicillin, amoxicillin-clavulanic acid, cefotaxime, imipenem, chloramphenicol, tetracycline, co-trimoxazole, nalidixic acid, ciprofloxacin, azithromycin, and rifaximin were determined. All genes encoding resistance determinants were detected by PCR or PCR plus DNA sequencing. The epidemiology of selected EAEC and ETEC strains was studied using multilocus sequence typing (MLST). The resistance to quinolones of EAEC and ETEC strains causing TD has significantly increased over the last decades, and high percentages have been found especially in patients traveling to India and sub-Saharan Africa. Sequence type 38 (ST38) and ST131, carrying the blaCTX-M-15 and blaCTX-M-27 genes, respectively, are highly prevalent among extended-spectrum β-lactamase (ESBL)-producing EAEC and ETEC strains. The cephamycinase ACT-20 is described in the present study for the first time in EAEC and ETEC strains causing TD in patients who had traveled to Central America. The percentages of resistance to azithromycin in EAEC and ETEC isolates from patients to Southeast Asia/India and Africa are above 25%. Meanwhile, rifaximin is still active against EAEC and ETEC, with the prevalence of resistant strains not being high. In conclusion, fluoroquinolones should no longer be considered the drugs of choice for the prevention or treatment in TD for travelers traveling to India and Africa. Azithromycin and rifaximin are still a good alternative to treat TD caused by EAEC or ETEC.

Characterization of antimicrobial resistance genes in Haemophilus parasuis isolated from pigs in China.

BackgroundHaemophilus parasuis is a common porcine respiratory pathogen that causes high rates of morbidity and mortality in farmed swine. We performed a molecular characterization of antimicrobial resistance genes harbored by H. parasuis from pig farms in China.MethodsWe screened 143 H. parasuis isolates for antimicrobial susceptibility against six fluoroquinolone antibiotics testing by the broth microdilution method, and the presence of 64 antimicrobial resistance genes by PCR amplification and DNA sequence analysis. We determined quinolone resistance determining region mutations of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). The genetic relatedness among the strains was analyzed by pulsed-field gel electrophoresis.ResultsSusceptibility test showed that all isolates were low resistance to lomefloxacin (28.67%), levofloxacin (20.28%), norfloxacin (22.38%), ciprofloxacin (23.78%), however, high resistance levels were found to nalidixic acid (82.52%) and enrofloxacin (55.94%). In addition, we found 14 antimicrobial resistance genes were present in these isolates, including blaTEM-1, blaROB-1, ermB, ermA, flor, catl, tetB, tetC, rmtB, rmtD, aadA1, aac(3′)-llc, sul1, and sul2 genes. Interestingly, one isolate carried five antibiotic resistance genes (tetB, tetC, flor, rmtB, sul1). The genes tetB, rmtB, and flor were the most prevalent resistance genes in H. parasuis in China. Alterations in the gyrA gene (S83F/Y, D87Y/N/H/G) were detected in 81% of the strains and parC mutations were often accompanied by a gyrA mutation. Pulsed-field gel electrophoresis typing revealed 51 unique patterns in the isolates carrying high-level antibiotic resistance genes, indicating considerable genetic diversity and suggesting that the genes were spread horizontally.DiscussionThe current study demonstrated that the high antibiotic resistance of H. parasuis in piglets is a combination of transferable antibiotic resistance genes and multiple target gene mutations. These data provide novel insights for the better understanding of the prevalence and epidemiology of antimicrobial resistance in H. parasuis.

Molecular characterization of antimicrobial resistance genes against Staphylococcus aureus isolates from Trinidad and Tobago

Staphylococcus aureus continues to pose major public health challenges in many areas because of antibiotic resistance problems. In the Caribbean, especially Trinidad and Tobago, the challenge is not different. This study was performed to evaluate the antimicrobial resistance gene prevalence among S. aureus isolates in Trinidad and Tobago. Standard and molecular microbiological methods, including the Microscan automated system, DNA microarray and multi locus sequence typing (MLST) analysis, were performed on 309 clinical S. aureus isolates recovered from patients who were treated at three of the country's main health institutions. S. aureus exhibited susceptibilities ≥80% to eleven of the 19 antimicrobials tested against it, and these belong to the most commonly used and available antibiotics in the country. While the antibiotic to which it was most susceptible of the commonly used antibiotics was trimethoprim/sulfamethoxazole, the antibiotics to which it was least susceptible or most resistant to were ampicillin and penicillin. S. aureus isolates from the pediatric ward produced the greatest rate of susceptibility among the isolates recovered from patients admitted into hospitals, while isolates from Accident and Emergency rooms displayed the greatest susceptibilities among patients from the community. S. aureus isolates from the country did not harbor acquired resistant genes targeting clindamycin/macrolides (ermB), linezolid (cfr) or vancomycin (vanA). The blaZ gene, which is the most common beta lactam (Penicillinase) resistance mechanism for S. aureus, was observed in 88.7% of the methicillin susceptible S. aureus, while methicillin resistance mediated by the mec gene was present in 13.6%. Most of the resistance markers found in MRSA isolates were significantly associated with the ST239-MRSA-III strain in this study, and all isolates that belonged to the USA300 strain, which additionally encoded both the PVL gene and ACME cluster, belonged to CC8. Several resistant genes, such as vanA, cfr and ermB, mediating resistance in S. aureus, are currently non-existent in Trinidad and Tobago. However, the majority of SCCmec genes were observed, suggesting that there is ongoing nosocomial transmission with minimal community transmission. This calls for stringent antibiotic stewardship and policies in the country.

Longitudinal characterization of antimicrobial resistance genes in feces shed from cattle fed different subtherapeutic antibiotics

BackgroundEnvironmental transmission of antimicrobial-resistant bacteria and resistance gene determinants originating from livestock is affected by their persistence in agricultural-related matrices. This study investigated the effects of administering subtherapeutic concentrations of antimicrobials to beef cattle on the abundance and persistence of resistance genes within the microbial community of fecal deposits. Cattle (three pens per treatment, 10 steers per pen) were administered chlortetracycline, chlortetracycline plus sulfamethazine, tylosin, or no antimicrobials (control). Model fecal deposits (n = 3) were prepared by mixing fresh feces from each pen into a single composite sample. Real-time PCR was used to measure concentrations of tet, sul and erm resistance genes in DNA extracted from composites over 175 days of environmental exposure in the field. The microbial communities were analyzed by quantification and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S-rRNA.ResultsThe concentrations of 16S-rRNA in feces were similar across treatments and increased by day 56, declining thereafter. DGGE profiles of 16S-rRNA differed amongst treatments and with time, illustrating temporal shifts in microbial communities. All measured resistance gene determinants were quantifiable in feces after 175 days. Antimicrobial treatment differentially affected the abundance of certain resistance genes but generally not their persistence. In the first 56 days, concentrations of tet(B), tet(C), sul1, sul2, erm(A) tended to increase, and decline thereafter, whereas tet(M) and tet(W) gradually declined over 175 days. At day 7, the concentration of erm(X) was greatest in feces from cattle fed tylosin, compared to all other treatments.ConclusionThe abundance of genes coding for antimicrobial resistance in bovine feces can be affected by inclusion of antibiotics in the feed. Resistance genes can persist in feces from cattle beyond 175 days with concentrations of some genes increasing with time. Management practices that accelerate DNA degradation such as frequent land application or composting of manure may reduce the extent to which bovine feces serves as a reservoir of antimicrobial resistance.

Characterization of antimicrobial resistant Salmonella enterica serovars Enteritidis and Typhimurium isolates from animal and food in Southern Italy

In the last two decades, the emergence and spread of antimicrobial-resistant pathogens, among them Salmonella, has become a serious health hazard worldwide, and specifically the high incidence of multidrug resistance has been encountered widely in many European countries. This study examines the antimicrobial susceptibility of Salmonella enterica strains Typhimurium and Enteritidis isolated in Campania and Calabria region (Southern Italy) from animal and food of animal origin. The relationship of antibiotic resistance phenotype and the presence of some resistance genes has been also investigated. As expected, our results show that resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides and tetracycline is common, although resistance to other antibiotics (i.e.: nalidixic acid) and other resistance patterns occur. The genetic resistant patterns have been partially described for this food-borne pathogen but efforts are needed to realize the complete characterization of antimicrobial resistance genes.