MLSB Resistance Research Articles

Resistant S. aureus Isolates Capable of Producing Biofilm from the Milk of Dairy Cows with Subclinical Mastitis in Slovakia

Staphylococcus spp. is the most common cause of mastitis, with a significantly low cure rate. Bacterial characteristics like adhesion and biofilm formation, as well as extracellular factors, can affect the pathogenesis of staphylococcal mastitis. The study’s objectives were to confirm S. aureus, assess their antibiotic resistance, identify methicillin resistance genes, verify biofilm formation, and detect biofilm-associated genes from bovine mastitis samples using multiplex PCR (mPCR). From 215 milk samples, six were confirmed as S. aureus. Most isolates were sensitive to all measured antibiotics. One isolate was identified as an inducible form of MLSB resistance (macrolides, lincosamides, and streptogramin B resistance), while the other two isolates were resistant to penicillins and carboxypenicillins. In S. aureus cultures used for methicillin resistance genotypic analysis by PCR, the mecA and mecC genes were not found. Biofilm formation phenotypes were determined in four strains. An mPCR analysis revealed that all strains of S. aureus carried icaABCD, agrA, srtA, fnbA, clfA, and clfB genes. Only in one isolate was the fnbB gene detected; the bap gene was not detected in any of the isolates. This emphasizes the importance of using appropriate treatment and continuous monitoring of S. aureus to prevent the spread of antibiotic-resistant strains in dairy cow farms.

Novel tetracycline resistance gene tet(65) located on a multi-resistance Corynebacterium plasmid.

Corynebacterium (C.) sp. 22KM0430 related to C. oculi and isolated from a dog exhibited resistance to tetracycline, and its WGS analysis revealed a putative resistance gene on a 35 562-bp plasmid also harbouring the MLSB resistance gene erm(X). To characterize the novel tetracycline resistance gene tet(65) and demonstrate its functionality by expression in C. glutamicum and Escherichia coli and plasmid curing of the host strain. tet(65) was cloned with and without its repressor tetR(65) and expressed in C. glutamicum DSM20300 and E. coli DH5α. Plasmid was cured by non-selective passages. Minimal inhibitory concentrations (MICs) of tetracyclines were determined according to CLSI guidelines. Association of tet(65) with efflux was shown by the addition of reserpine to MIC assays. Phylogenetic position and transmembrane structure of Tet(65) were analysed using MEGA11 and DeepTMHMM. Tet(65) shows 73% amino acid identity with the closest related Tet(Z), contains 12 transmembrane domains and is structurally related to the Major Facilitator Superfamily. The tetracycline MICs decreased in the plasmid-cured strain and increased when tet(65) was expressed in C. glutamicum and in E. coli. The MICs of tetracycline decreased in the presence of reserpine indicating that tet(65) functions as an efflux pump. A GenBank search also identified tet(65) in C. diphtheriae and Brevibacterium (B.) casei and B. luteolum. A novel tetracycline efflux gene tet(65) was identified in a C. oculi related species and was also present in the human pathogen C. diphtheriae and in Brevibacterium species indicating broader potential for dissemination.

Transfer and accumulation of antibiotic resistance genes and bacterial pathogens in the mice gut due to consumption of organic foods

Over the last few decades, organic food demand has grown largely because of increasing personal health concerns. Organic farming introduces antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) into foods. However, potential effects of organic foods on the gut microbiome and ARGs have been overlooked. Using high-throughput quantitative PCR and 16S rRNA high-throughput sequencing technology, we examined 132 ARGs from major classes, eight transposase genes, universal class I integron-integrase gene (intI), clinical class I integron-integrase gene (cintI), and the bacterial community in mouse gut after 8 weeks with an either organic or inorganic lettuce and wheat diet. A total of 8 types of major ARGs and 10 mobile genetic elements (MGEs) were detected in mice gut, including tetracycline, multidrug, sulfonamide, aminoglycoside, beta-lactamase, chloramphenicol, MLSB and vancomycin resistance genes. We found that abundance and diversity of ARGs, mobile gene elements, and potential ARB in the gut increased with time after consumption of organic foods, whereas no significant changes were observed in inorganic treated groups. Moreover, MGEs, including IS613, Tp614 and tnpA_03 were found to play an important role in regulating ARG profiles in the gut microbiome following consumption of organic foods. Importantly, feeding organic food increased the relative abundance of the potentially antibiotic-resistant pathogens, Bacteroides and Streptococcus. Our results confirm that there is an increasing risk of ARGs and ARB in the gut microbiome, which highlights the importance of organic food industries taking into account the potential accumulation and transmission of ARGs as a risk factor.

Nasal carriage of methicillin and inducible clindamycin resistant Staphylococcus aureus among healthcare workers in a tertiary care hospital, Kathmandu, Nepal

Introduction and Objectives: Transmission of methicillin-resistant Staphylococcus aureus (MRSA) from healthcare workers is one of the most frequent causes of nosocomial infections globally. There is a significant burden of nosocomial MRSA infections in low and low-middle income countries (LMICs), including Nepal. The present study investigated the rate of nasal carriage of MRSA among the healthcare workers in a tertiary care hospital, in Kathmandu, Nepal with emphasis on inducible macrolide-lincosamide-streptogramin B (iMLSB) resistance. Material and method: The study was conducted at Star Hospital, Lalitpur, Nepal, from September 2022 to November 2022. Healthcare workers (HCWs) working at the diffeerent departments of the hospital were enrolled. Nasal swabs from both anterior nares of HCWs were collected aseptically and cultured on Mannitol Salt agar. S. aureus was identified by Gram stain and standard biochemical tests. Antibiotic susceptibility of S. aureus was performed by disc diffusion method. MRSA isolates were detected phenotypically by disc diffusion method using cefoxitin disc (30 µg), and inducible clindamycin resistance was detected phenotypically by the D-zone test. Results: Total 105 HCWs were enrolled in the study. Out of 105 HCWs, 14 (13.3%) were positive for S. aureus among which 6 (5.7%) were MRSA carriers. The nasal carriage of MRSA was highest among doctors (16.7%) and the HCWs of the post-operative department (14.3%). All the isolated MRSA were susceptible to chloramphenicol and vancomycin. Inducible MLSB resistance was detected in 33.3% MRSA while the rate was 21.4% in all isolated S. aureus. Conclusion: The study demonstrated that HCWs could be the potential source of nosocomial infection by methicillin and inducible clindamycin resistant S. aureus. Thus, preventive measures should be initiated to mitigate the risk of its spread and the test for detection of inducible clindamycin resistance should be incorporated into the routine antibiotic susceptibility testing in hospital settings. IMC J Med Sci. 2024; 18(1):005. DOI: https://doi.org/10.55010/imcjms.18.005 *Correspondence: Gaurab Pandey, Non-Communicable Disease Laboratory, National Public Health Laboratory, Teku, Kathmandu, 44600, Nepal; E-mail: pandeygaurab67@gmail.com

Prevalence of erythromycin-resistant emm92-type invasive group A streptococcal infections among injection drug users in West Virginia, United States, 2021-23.

Increasing incidence of invasive group A Streptococcus (iGAS) disease has been reported in Europe and the USA over the past several years. Coupled with this are observations of higher rates of resistance to erythromycin and clindamycin. To characterize iGAS and pharyngitis isolates from West Virginia (WV), a US state outside of the national Active Bacteria Core surveillance purview, where risk factors associated with iGAS infections are prevalent. Seventy-seven invasive group A Streptococcus isolates were collected from 67 unique patients at the J.W. Ruby Memorial Hospital Clinical Microbiology Laboratory in WV from 2021 to 2023. Invasive isolates and 20 unique pharyngitis isolates were tested for clindamycin and erythromycin susceptibility in the clinical laboratory. Patient demographic and clinical information was retrieved from patient electronic health records. Isolates were further characterized based on emm subtype and detection of MLSB resistance determinants. Twenty-six (39%) isolates were of a single emm92 type. All emm92 isolates were uniformly erythromycin/clindamycin resistant with inducible or constitutive MLSB resistance imparted by the plasmid-borne erm(T) gene. The majority of emm92 infections were associated with adult patients who reported IV drug use, whereas no pharyngitis infections were caused by an emm92 strain. Overall, 51 (76%) of the 67 iGAS isolates were determined to carry MLSB resistance. Isolates of emm92 type (clonal subtype emm92.0) were associated with iGAS infections in adult IV drug users, but not with paediatric pharyngitis, and were uniformly resistant to erythromycin and clindamycin.

Effects of soil solarization combined with manure-amended on soil ARGs and microbial communities during summer fallow

Soil solarization (SS) is a technique for managing pathogens and weeds, which involves covering with transparent plastic to increase soil temperature during summer fallow (SF). However, SS also alters the diversity of bacterial communities. Therefore, during SF, various organic modifiers are used in combination with SS to improve its efficacy. Organic amendments may contain antibiotic resistance genes (ARGs). Greenhouse vegetable production (GVP) soils are vital to ensure food security and ecological balance. However, comprehensive study on the effects of SS combined with different types of manure on ARGs in GVP soils during SF remains unclear. Therefore, this study employed high-throughput qPCR to explore the effects of different organic amendments combined with SS on the abundance changes of ARGs and mobile genetic elements (MGEs) in GVP soils during SF. The abundance and diversity of ARGs and MGEs in GVP soils with different manure fertilization and SS decreased during SF. Horizontal gene transfer via MGEs (especially integrases 45.80%) induced by changes in environmental factors (NO3−-N 14.7% and NH4+-N) was the main factor responsible for the changes in ARGs. Proteobacteria (14.3%) and Firmicutes were the main potential hosts of ARGs. Network analysis suggested that Ornithinimicrobium, Idiomarina and Corynebacterium had positive correlations with aminoglycosides, MLSB, and tetracycline resistance genes. These results provide new insights to understand the fate of ARGs in the GVP soils by manure-amended combined with SS during SF, which may help to reduce the spread of ARGs.

Tn5406, a staphylococcal transposon associated with macrolide-lincosamide-streptograminb resistance in clinical isolates of Staphylococcus aureus.

In this study, we aimed to investigate the occurrence of MLSb resistance in clinical isolates of Staphylococcus aureus with respect to their association with transposons. The present study was performed with clinical isolates of S.aureus. The MLSb resistant phenotypes in the obtained isolates were determined by D zone test or double disc diffusion test as per CLSI 2020 guidelines. MLSb resistance encoding genes were detected by PCR. The genes tested were ermA, ermB, ermC, msrA, mphC, vga, vgb and lnuB. The MLSb resistant Staphylococcal isolates were selected to analyze the association of the genes with mobile genetic elements Tn554, Tn5406, Tn917, Tn6133, Tn551 by PCR based method. Primer pairs were designed using sequences from transposons and the resistance genes, respectively. During this study, 268 isolates of S.aureus were obtained of which 233 (86.94%) isolates exhibited different MLSb resistant phenotypes. The predominant gene among the MLSb resistant isolates was msrA followed by vgaA and mphC genes. PCR assay was employed to determine whether the genes msrA, mphC and vgaA were carried by Tn554, Tn5406, Tn917, Tn6133, Tn551 transposons. PCR amplification with the designed primer pairs revealed vgaA gene being part of Tn5406. The presence of Tn5406 in all the vgaA harboring isolates highlights its potential of spread across isolates. Moreover, the co-existence of different MLSb resistance encoding genes observed in the study shows that the combination of genes involved in different mechanism mediated the nature of MLSb resistance.

Removal behavior and key drivers of antibiotic resistance genes in two full-scale leachate treatment plants

Leachate is a critical reservoir of antibiotic resistance genes (ARGs) and its proper treatment is closely related to human health and ecosystem safety. Here, we used high-throughput qPCR to explore the removal behavior of ARGs in two full-scale leachate treatment plants (LTPs) where biological treatment and membrane filtration processes were integrated. A total of 286 ARGs and 55 mobile genetic elements (MGEs) were detected, with aminoglycoside, multidrug and MLSB resistance genes being the most prevalent and abundant. Anaerobic digestion was found to be an important pretreatment process for leachate, while anoxic/aerobic tanks in membrane bioreactor (MBR) acted as incubators for ARGs due to their significant proliferation effect on ARGs. Integrated membrane filtration (UF-NF-RO) excelled in ARGs removal with absolute abundances reduced by 3 to 6 orders of magnitude, from about 109 copies/mL in raw leachate to 103-105 copies/mL in effluents. Our results also showed that leachate treatment processes significantly altered the composition of ARGs and bacterial communities. Procrustes analysis and network analysis revealed strong associations between microbes and ARGs, with several hub genes and bacterial genera identified. Structural equation models (SEMs) indicated that bacterial composition, MGEs and basic water properties were the key drivers shaping ARGs dynamics in the raw leachate, biological system and filtration system, respectively. Notably, several pathogens (e.g., Klebsiella, Vibrio, Aeromonas) were closely correlated with ARGs in raw leachate and may amplify the dissemination risks of ARGs. Moreover, insertion sequences in biological systems would accelerate the horizontal gene transfer of ARGs. In short, this study provides new insights into the mechanisms of ARGs removal and dissemination behavior in industrial-scale LTPs.

Phenotypic and Genotypic Characterization of Macrolide, Lincosamide and Streptogramin B Resistance among Clinical Methicillin-Resistant Staphylococcus aureus Isolates in Chile.

Macrolides, lincosamides, and type B streptogramins (MLSB) are important therapeutic options to treat methicillin-resistant Staphylococcus aureus (MRSA) infections; however, resistance to these antibiotics has been emerging. In Chile, data on the MLSB resistance phenotypes are scarce in both community-(CA) and hospital-acquired (HA) MRSA isolates. Antimicrobial susceptibility to MLSB was determined for sixty-eight non-repetitive isolates of each HA-(32) and CA-MRSA (36). Detection of SCCmec elements, ermA, ermB, ermC, and msrA genes was performed by PCR. The predominant clones were SCCmec I-ST5 (HA-MRSA) and type IVc-ST8 (CA-MRSA). Most of the HA-MRSA isolates (97%) showed resistance to clindamycin, erythromycin, azithromycin, and clarithromycin. Among CA-MRSA isolates, 28% were resistant to erythromycin, azithromycin, and 25% to clarithromycin. All isolates were susceptible to linezolid, vancomycin, daptomycin and trimethoprim/sulfamethoxazole, and over 97% to rifampicin. The ermA gene was amplified in 88% of HA-MRSA and 17% of CA-MRSA isolates (p < 0.001). The ermC gene was detected in 6% of HA-SARM and none of CA-SARM isolates, whereas the msrA gene was only amplified in 22% of CA-MRSA (p < 0.005). Our results demonstrate the prevalence of the cMLSB resistance phenotype in all HA-MRSA isolates in Chile, with the ermA being the predominant gene identified among these isolates.

Ferric chloride further simplified the horizontal gene transfer network of antibiotic resistance genes in anaerobic digestion

The role of ferric chloride (FC) on the reduction of antibiotic resistance genes (ARGs) in anaerobic digestion (AD) system was investigated from the perspective of vertical (VGT) and horizontal gene transfer (HGT) network through the high-throughput qPCR (HT-qPCR). Although FC showed limited impacts on methane production in AD of swine manure, the tetracycline and MLSB resistance genes were specifically reduced at the end, where tetQ of antibiotic target protection and ermF of antibiotic target alteration contributed the most to the reduction. Both VGT and HGT network were divided into three modules, and the complexity of HGT network was largely reduced along with AD, where the HGT connection was reduced from 683 (Module III) to 172 (Module I), and FC addition could further reduce the relative abundance of ARG hosts in Module I. The contribution of VGT and HGT to the changes of ARGs in AD was further deciphered, and although the VGT reflected by the changes of microbial community contributed the most to the dynamics of ARGs (68.0 %), the HGT contribution could further be reduced by the FC addition. This study provided a new perspective on the fate of ARGs response to the FC addition in the AD system.

Dispersal of antibiotic resistance genes in an agricultural influenced multi-branch river network

Rivers in agricultural regions serve as an important sink for livestock and poultry farm runoff, fertilizer runoff, and country living sewage, which could bring antibiotic resistance genes (ARGs) contaminations. However, the diversity and distribution of ARGs has not been well documented in the agricultural influenced river. Here, the diversity of ARGs, and their relationship with biochemical factors were determined in the surface water in an agricultural region of the Jialing River and its five rural branches. The 218 unique ARGs encoding resistance to eight major antibiotic classes have been detected using high-throughput quantitative PCR. The branches of the river had a remarkably higher abundance of ARGs than the mainstream. The aminoglycoside, beta_Lactamase, MLSB, and Multidrug resistance genes were significantly enriched in the branches compared to the mainstream. Compared with the mainstream, the ARGs profiles in the branches showed obvious higher spatial variability. Significant correlation between ARGs profiles and bacterial community structures were observed, and network analysis further showed that the ARGs were associated with their potential hosts, such as Ottowia and Novosphingobium. Redundancy discrimination analysis revealed that Cu content has a significant contribution to the increase of ARGs in the river. The microbial diversity index was negatively correlated with the abundance of the ARGs. These results provide evidence for the enrichment of ARGs in the agricultural influenced river and branches due to the joint influence of chemical and microbial variables.

Pollution Characteristics and Driving Factors of Antibiotic Resistance Genes in Dexing Copper Mine

Environmental antibiotic resistance genes (ARGs) are a type of emerging pollutant that has been widely concerning. However, investigations into the contamination of ARGs in mining areas have been scarce. Here, the types, abundances, and influencing factors of ARGs and mobile genetic elements (MGEs) were investigated in soil/sediment of the Dexing copper mine area in June 2019 by using high-throughput quantitative polymerase chain reaction (HT-qPCR). Furthermore, the influence of heavy metals and MGEs factors on ARGs was studied using the multivariate statistical analysis method. The results showed that there were a variety of ARGs in the Dexing copper mining area, and the maximum detected number of ARGs was 70. At the relative abundance level, the relative abundance of individual sites reached 0.085. In the Dexing copper mine, multidrug, MLSB, β-lactamases, tetracycline, and aminoglycoside resistance genes were the dominant ARG classes based on their numbers. The efflux pump was the most dominant resistance mechanism, followed by antibiotic deactivation and cellular protection. There was a significant positive correlation between the abundance of ARGs and MGEs (P<0.05), and TnpA04 and Inti1 were the most important MEGs in Dexing copper mine samples, indicating that horizontal gene transfer might be an important mechanism for the spread of environmental ARGs. The results of Pearson correlation analysis and RDA analysis showed that the content of Cu was significantly positively correlated with the detected numbers and abundance of ARGs (P<0.05), suggesting that the high content of Cu in the Dexing copper mining area might be an important driving factor for the formation of ARGs.

Antibiotics Susceptibility Profile of &amp;lt;i&amp;gt;Staphylococcus aureus&amp;lt;/i&amp;gt; Clinical Isolates Collected in Hospitals in the City of Douala (Cameroon)

Introduction: Staphylococcus aureus is one of the most important agents involved in community and hospital-acquired infections. Due to the multi-drug resistance of strains to antibiotics, treatment to eradicate it is becoming more difficult and poses a global public health problem. Methodology: This was a cross-sectional study conducted from March to August 2020 in hospitals in the city of Douala, including all S. aureus isolates from diagnostic samples. Strain identification and antibiotic susceptibility testing were performed using the Vitek2 CompactTM (BioMerieux). Results: During the study period, 136 non-repetitive S. aureus strains were identified with a high frequency of methicillin-resistant S. aureus of 78.7%. The majority of the strains originated from the Douala General Hospital (66.9%) and was most frequently isolated from blood culture samples (55.1%). The study of biochemical characteristics showed that most of the strains identified had between 87% and 99% homology with the reference strain. The most active antibiotics were Quinupristin/Dalfopristin (94.2%), Linezolid (87.8%) and Vancomycin (84.2%). Methicillin resistance was associated with decreased susceptibility of S. aureus to other antibiotics such as Gentamycin (44.9%), Erythromycin (38.2%), Tetracycline (38.3%), Trimethoprim (21.4%), Ciprofloxacin (19.1%) and Levofloxacin (24.0%). Inducible MLSb and constitutive resistance phenotypes were identified with 26.7% and 22.8% respectively. Conclusion: The sensitivity of S. aureus strains differs from one antibiotic family to another, and remains good for molecules that are not available in our context. The high frequency of Methicillin-Resistant S. aureus shows the continuous progression of multi-resistant strains of S. aureus and their decreased sensitivity to usual antibiotics becomes more and more alarming.

Determination of nasal carriage and skin colonization, antimicrobial susceptibility and genetic relatedness of Staphylococcus aureus isolated from patients with atopic dermatitis in Szczecin, Poland

BackgroundAtopic dermatitis (AD) is one of the most frequent chronic and inflammatory skin condition. AD is characterized by damaged epidermal barrier, xerosis and pruritus of eczematous skin lesions which tend to flare. The duration and frequency of exacerbation of AD symptoms markedly affects the quality of patient life. AD results from the interplay between host genetics, immunity, and environmental factors, however the detailed pathogenesis of this disease is still not entirely cleared. Furthermore, disturbances of the skin microbiota and skin functional impairment predispose to secondary skin infections. Staphylococcus aureus colonizes skin and mucous membranes of 20 to 80% of healthy individuals and of 90% of patients with AD in whom this bacterium is accounted as an important AD exacerbating factor. It is also proven, that S. aureus nasal carriage significantly increases the risk for self-transmission and endogenous infection. In the current study the presence of S. aureus either in nasal vestibule and on lesioned skin of 64 patients with AD enrolled in 10-year autovaccination program was determined. The genetic relatedness of 86 S. aureus isolated from patients nose and skin using Pulsed Field Gel Electrophoresis (PFGE) and antimicrobial susceptibility of all strains to methicillin, erythromycin, clindamycin, mupirocin, gentamicin, amikacin, tetracycline, chloramphenicol and cotrimoxazole was also evaluated.ResultsIn total 23 PFGE genotypes and 24 unique patterns were distinguished. 34 patients were S. aureus nasal carriers. Simultaneous presence of S. aureus in nose and on affected skin was found in 16 carriers colonized by indistinguishable or potentially related S. aureus vs 2 carriers colonized with non-related S. aureus in nasal vestibule and on skin.4 isolates were methicillin resistant (MRSA) among which 3 showed constitutive MLSB resistance phenotype and remaining one was resistant to tetracycline and chloramphenicol.In 4 isolates inducible MLSB resistance phenotype was found, one of them was additionally resistant to tetracycline. 7 S. aureus were mupirocin resistant among them 3 - isolated from one patient, were resistant simultaneously to tetracyclines and chloramphenicol. 7 strains demonstrated resistance to chloramphenicol and susceptibility to all tested antimicrobial agents. The susceptibility to gentamicin, amikacin and cotrimoxazole among all examined S. aureus was confirmed.ConclusionThe obtained results indicated non-clonal structure of S. aureus circulating in AD patients. PFGE results showed the clonal-structure of vast majority of S. aureus isolated from nose and skin from nasal carriers what may prove the autoinfection in these patients. All examined patients the moderate or strong severity of AD was reported. Susceptibility to most antibiotics among isolated strains was also observed.

Have Methicillin Resistant Staphylococcus aureus clinical isolates to be also resistant to Streptogramins?

Background: Methicillin Resistant Staphylococcus aureus (MRSA) is a significant pathogen causing high morbidity and mortality. The extensive misuse of antibiotics has led to the willing of using older compounds like macrolide, lincosamides, and streptogramin (MLS) antimicrobials family. This study aimed to detect phenotypic and molecular characterization of macrolide, lincosamides, and streptogramins resistance in MRSA clinical isolates.Methods: Antimicrobial susceptibility testing of 50 MRSA clinical isolates to MLS agents and quinupristin-dalfopristin (Q -D) was performed using disk diffusion method. Besides, polymerase chain reaction was conducted for amplification of genes related to streptogramins resistance (ermA, ermB, ermC, msrA, vatA, vatB, vatC, vgaA and vgbA).Results: MLSB resistance phenotypes detected were cMLSB 9(18%), iMLSB 5(10%), MSB 8(16%), and 3(6%) isolates were LSA phenotype. No resistance to Q-D was detected in any of the tested isolates .The most prevalent MLSB resistance genes were ermC in the cMLSB and msrA in the MSB. The 3 (6%) LSA phenotype isolates expected to be SA resistant, and the 22(44%) isolates expected to be SB resistant, were sensitive to Q-D. Genes related to Q-D resistance (vatA, vatB, vatC, vgaA and vgbA genes) were not detected confirming the susceptibility of all the tested isolates to Q-D by disk diffusion method. . Conclusion: Accurate identification of phenotypic and genotypic MLSB resistance is a crucial approach to decrease the antibiotic resistance rates. The study revealed a high prevalence of the cMLSB phenotype and the most prevalent resistance determinants was ermC.

Spatial and temporal dynamics of microbiomes and resistomes in broiler litter stockpiles

Farmers apply broiler chicken litter to soils to enrich organic matter and provide crops with nutrients, following varying periods of stockpiling. However, litter frequently harbors fecal-derived microbial pathogens and associated antibiotic resistance genes (ARGs), and may be a source of microbial contamination of produce. We coupled a cutting-edge Loop Genomics long-read 16S rRNA amplicon-sequencing platform with high-throughput qPCR that targeted a suite of ARGs, to assess temporal (five time points over a 60-day period) and spatial (top, middle and bottom layers) microbiome and resistome dynamics in a broiler litter stockpile. We focused on potentially pathogenic species from the Enterobacteriaceae, Enterococcaceae and Staphylococcaceae families associated with food-borne disease. Bacterial diversity was significantly lower in the middle of the stockpile, where targeted pathogens were lowest and Bacillaceae were abundant. E. coli was the most abundant Enterobacteriaceae species, and high levels of the opportunistic pathogen Enterococcus faecium were detected. Correlation analyses revealed that the latter was significantly associated with aminoglycoside (aac(6′)-Ib(aka aacA4), aadA5), tetracycline (tetG), vancomycin (vanC), phenicol (floR) and MLSB (mphB) resistance genes. Staphylococcaceae were primarily non-pathogenic, but extremely low levels of the opportunistic pathogen S. aureus were detected, as was the opportunistic pathogen S. saprophyticus, which was linked to vancomycin (vanSA, vanC1), MLSB (vatE, ermB) and tetracycline (tetK) resistance genes. Collectively, we found that stockpile microbiomes and resistomes are strongly dictated by temporal fluctuations and spatial heterogeneity. Insights from this study can be exploited to improve stockpile management practice to support sustainable antimicrobial resistance mitigation policies in the future.

Genomic basis of antimicrobial resistance in non-toxigenic Clostridium difficile in Southeast Asia

Despite being incapable of causing Clostridium difficile infection, non-toxigenic C. difficile (NTCD) may still be relevant. This study explored the role of NTCD as a reservoir of accessory antimicrobial resistance (AMR) genes in NTCD from Southeast Asia. This region has high rates of antimicrobial use, a high prevalence of NTCD and phenotypic AMR in such strains. More than half of the 28 NTCD strains investigated had at least one accessory AMR gene on mobile genetic elements (MGEs) which were similar to the elements found in other bacteria, including Erysipelothrix rhusiopathiae and Streptococcus suis, both of which are found in the pig gut. Thus, C. difficile may facilitate the movement of AMR genes between different hosts within a wide range of pathogenic bacteria. C. difficile β-lactamases were not located on MGEs and were unlikely to be transferred. Concordance between the MLSB resistance genotype and phenotype was low, suggesting multiple resistance mechanisms, many of which remain unknown. On the contrary, there was a high concordance between resistance genotype and phenotype for both fluoroquinolones and rifaximin. From an epidemiological perspective, NTCD populations in Southeast Asia comprised members of evolutionary clades 1 and 4, which are thought to have originated from Europe and Asia, respectively. This population structure reflects the close relationship between the people of the two regions.

Molecular Characterization, Drug Resistance and Virulence Analysis of Constitutive and Inducible Clindamycin Resistance Staphylococcus aureus Strains Recovered from Clinical Samples, Tehran-Iran.

BackgroundMacrolide-lincosamide streptogramin B family is one of the important alternative antibiotics for treating staphylococcal infections. The aim of this study was to determine the characteristics and prevalence of antibiotic resistance genes in different coagulase types of clinical Staphylococcus aureus strains.MethodsIn the present study, 86 isolates with different phenotypes of MLSB resistance were investigated. In vitro susceptibility was assessed by the disk diffusion and broth microdilution methods. PCR assays were used to detect resistance-related genes. Coagulase and SCCmec types were identified by multiplex PCR assay.ResultsThe prevalences of constitutive MLSB, inducible MLSB, and MS phenotypes were found to be 23%, 14.2%, and 4.9%, respectively. The rates of resistance to mupirocin, fusidic acid, and tigecycline were found to be 9.3%, 4.6%, and 2.3%, respectively. The top three predominant resistance genes were mecA, tet(M), erm(C) representing 75.6, 50, and 40.7% of isolates. mupA (7%), fusB (3.5%), and fusC (1.2%) genes were also detected among tested isolates. Coagulase types were mainly type II (34.9%), followed by III (32.6%), V (20.9%), and I (11.6%).ConclusionThese findings indicated high resistance rate and low genetic variability with the prominence of coa type II, highlighting the particular importance of diagnosis of these strains to avoid treatment failure.

Detection of Clindamycin Resistance Antibiotic Genes among Staphylococcus Isolates by Using Real Time PCR

STAPHYLOCOCCUS aureus is the most common pathogens and an important nosocomial pathogen. This bacterium has ability to acquire resistance to different antibiotic classes. Erythromycin and clindamycin have been used for treating skin and soft-tissue infections caused by these bacteria. Although, expression of macrolide-lincosamide-streptogramin B resistance (MLSB) can limit the effect of these drugs.This study aimed to detect the presence of genes encoding resistance to macrolides, lincosamides and streptogramins (MLSB) among clinical Staphylococcus strains.A total of 100 Staphylococcus isolates from clinical specimens (blood, sputum and wound) were collected. All these isolates were identified biochemically. They were tested against different antibiotics and double-disk diffusion method and genes were detected using PCR.Results show that 63 isolates were genotypically tested by using real time PCR for detection of Erm (B) and Erm (C) genes. (EryS ClinS) was detected in 50 (50%) of the isolates followed by constitutive phenotype of MLSB resistance (EryR ClinR) 29 (29%) and inducible MLSB resistance (EryRClinInd) 17 (17%), while the MSB phenotype (EryR ClinS) 4(4%) was the least frequent. 51(80.9) isolates have Erm (B) gene positive 33(52.3) strains have Erm (C) gene positive.This study investigate that the double disk diffusion test is a suitable test for detection of staphylococci which are resistant to clindamycin and should be used as fast detection and help in treatment of patients. And thus, the screening procedure using Real time PCR is sensitive in detection of Erm genes which are responsible for inducible resistance to clindamycin.

Genetic diversity and antimicrobial resistance of Staphylococcus aureus from recurrent tonsillitis in children.

The aim of this study was to analyze the prevalence of Staphylococcus aureus in the tonsils of children subjected tonsillectomy due to recurrent tonsilitis and to determine the spa types of the pathogens, carriage of virulence genes and antimicrobial resistance profiles. The study included 73 tonsillectomized children. Bacteria, including S.aureus were isolated from tonsillar surface prior to tonsillectomy, recovered from tonsillar core at the time of the surgery, and from posterior pharynx 2-4weeks after the procedure. Staphylococcus aureus isolates were compared by spa typing, tested for antimicrobial susceptibility and for the presence of superantigenic toxin genes (sea-seu, eta, etb, tst, lukS/lukF-PV) by multiplex polymerase chain reaction. Seventy-three patients (mean 7.1±4.1years, 61.6% male) were assessed. The most commonly isolated bacteria were S.aureus. The largest proportion of staphylococcal isolates originated from tonsillar core (63%), followed by tonsillar surface (45.1%) and posterior pharynx in tonsillectomized children (18.2%, p=0.007). Five (6.3%) isolates were identified as MRSA (mecA-positive). Up to 67.5% of the isolates synthesized penicillinases (blaZ-positive isolates), and 8.8% displayed MLSB resistance. The superantigenic toxin genes were detected in more than half of examined isolates (56.3%). spa types t091, t084, and t002, and clonal complexes (CCs) CC7, CC45, and CC30 turned out to be most common. Staphylococcus aureus associated with RT in children showed pathogenicity potential and considerable genetic diversity, and no clones were found to be specific for this condition although further studies are needed.