scholarly journals Staphylococcus aureus in Intensive Pig Production in South Africa: Antibiotic Resistance, Virulence Determinants, and Clonality

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 317
Author(s):  
Ncomeka Sineke ◽  
Jonathan Asante ◽  
Daniel Gyamfi Amoako ◽  
Akebe Luther King Abia ◽  
Keith Perrett ◽  
...  
Keyword(s):  
South Africa ◽  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Meca Gene ◽  
Resistance Index ◽  
Control Measures ◽  
World Health ◽  
Diffusion Method ◽  
Multiple Antibiotic Resistance ◽  
Pig Production

Although Staphylococcus aureus is a major threat to the veterinary, agricultural, and public health sectors because of its zoonotic potential, studies on its molecular characterisation in intensive animal production are rare. We phenotypically and genotypically characterised antibiotic-resistant S. aureus in intensive pig production in South Africa, using the farm-to-fork approach. Samples (n = 461) were collected from the farm, transport vehicles, and the abattoir using the World Health Organisation on Integrated Surveillance of Antimicrobial Resistance (WHO-AGISAR) sampling protocol. Bacteria were isolated using selective media and identified using biochemical tests and polymerase chain reaction (PCR). Phenotypic resistance was determined using the disk diffusion method. Selected resistance and virulence genes were investigated using PCR. Clonality among the isolates was determined using the repetitive element sequence-PCR. In all, 333 presumptive staphylococcal isolates were obtained, with 141/333 (42.3%) identified as staphylococci biochemically. Ninety-seven (97; 68.8%) were confirmed as S. aureus using PCR, 52.6% of which were identified as methicillin-resistant S. aureus (MRSA) through the mecA gene. All the 97 S. aureus isolates (100%) were resistant to at least one of the antibiotics tested, with the highest resistance observed against erythromycin and clindamycin (84.50% each), and the lowest observed against amikacin (2.10%); 82.47% (80/97) were multidrug-resistant with an average multiple antibiotic resistance index of 0.50. Most of the phenotypically resistant isolates carried at least one of the corresponding resistance genes tested, ermC being the most detected. hla was the most detected virulence gene (38.14%) and etb was the least (1.03%). Genetic fingerprinting revealed diverse MRSA isolates along the farm-to-fork continuum, the major REP types consisting of isolates from different sources suggesting a potential transmission along the continuum. Resistance to antibiotics used as growth promoters was evidenced by the high prevalence of MDR isolates with elevated multiple antibiotic resistance indices >0.2, specifically at the farm, indicating exposure to high antibiotic use environments, necessitating antibiotic stewardship and proper infection control measures in pig husbandry and intensive pig production.

Evaluation of antibiotic resistance pattern and mecA gene frequency in methicillin-resistant Staphylococcus aureus strains collected from clinical specimens in Marand hospital and treatment centers, Iran

2021 ◽  
Author(s):  
Abolfazl Jafari-Sales ◽  
Zahra Sadeghi Deylamdeh ◽  
Afsoon Shariat
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Meca Gene ◽  
Disk Diffusion ◽  
Diffusion Method ◽  
Disk Diffusion Method ◽  
Methicillin Resistant ◽  
Medical Centers ◽  
Wide Range

Introduction: Staphylococcus aureus causes a wide range of infections and as a multivalent pathogen is one of the causative agents of nosocomial and community infections. Therefore, the aim of this study was to identify and determine the pattern of antibiotic resistance of methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients in hospitals and medical centers in Marand city and also to evaluate the presence of mecA gene. Materials and Methods: In this cross-sectional descriptive study, 385 samples of S. aureus were collected from different clinical samples of patients in hospitals and medical centers of Marand city. S. aureus was identified using standard biochemical methods.  Methicillin resistance was determined by disk diffusion method in the presence of oxacillin and cefoxitin. The pattern of antibiotic resistance of the strains was determined by disk diffusion method and according to CLSI recommendation and also PCR method was used to evaluate the frequency of MecA gene. Results: In the present study, out of 385 samples of S. aureus, 215 (55.84%) samples were methicillin resistant. PCR results for mecA gene showed that 110 samples had mecA gene.  The highest antibiotic resistance was observed against penicillin (100%) and erythromycin (83.63%). Most MRSA were isolated from urine and wound samples. Conclusion: The results of this study indicate the prevalence of methicillin-resistant species and also the increase in antibiotic resistance of MRSA to various antibiotics.  Therefore, in order to prevent increased resistance to other antibiotics, it is recommended to avoid inappropriate use of antibiotics.

scholarly journals Occurrence, Antimicrobial Resistance, and Molecular Characterization of Campylobacter spp. in Intensive Pig Production in South Africa

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 439
Author(s):  
Viwe Sithole ◽  
Daniel Gyamfi Amoako ◽  
Akebe Luther King Abia ◽  
Keith Perrett ◽  
Linda A. Bester ◽  
...  
Keyword(s):  
South Africa ◽  
Antibiotic Resistance ◽  
High Resistance ◽  
Virulence Genes ◽  
Diarrheal Disease ◽  
Efflux Pump ◽  
Diffusion Method ◽  
23S Rrna ◽  
Pig Production ◽  
Campylobacter Spp

Campylobacter spp. are among the leading foodborne pathogens, causing campylobacteriosis, a zoonotic infection that results in bacterial gastroenteritis and diarrheal disease in animals and humans. This study investigated the molecular epidemiology of antibiotic-resistant Campylobacter spp. isolated across the farm-to-fork-continuum in an intensive pig production system in South Africa. Following ethical approval, samples were collected over sixteen weeks from selected critical points (farm, transport, abattoir, and retail) using a farm-to-fork sampling approach according to WHO-AGISAR guidelines. Overall, 520 samples were investigated for the presence of Campylobacter spp., which were putatively identified using selective media with identity and speciation confirmed by polymerase chain reaction (PCR) of specific genes. Resistance profiles were ascertained by the Kirby–Bauer disk diffusion method. Antibiotic resistance and virulence genes were identified using PCR and DNA sequencing. Clonal relatedness was determined using ERIC-PCR. Altogether, 378/520 (72.7%) samples were positive for Campylobacter spp., with Campylobacter coli being the predominant species (73.3%), followed by Campylobacter jejuni (17.7%); 8.9% of the isolates were classified as “other spp”. Relatively high resistance was observed in C. coli and C. jejuni to erythromycin (89% and 99%), streptomycin (87% and 93%), tetracycline (82% and 96%), ampicillin (69% and 85%), and ciprofloxacin (53% and 67%), respectively. Multidrug resistance (MDR) was noted in 330 of the 378 (87.3%) isolates. The antibiotic resistance genes observed were tetO (74.6%), blaOXA-61 (2.9%), and cmeB (11.1%), accounting for the resistance to tetracycline and ampicillin. The membrane efflux pump (cmeB), conferring resistance to multiple antibiotics, was also detected in most resistant isolates. Chromosomal mutations in gyrA (Thr-86-Ile) and 23S rRNA (A2075G and A2074C) genes, conferring quinolone and erythromycin resistance, respectively, were also found. Of the virulence genes tested, ciaB, dnaJ, pldA, cdtA, cdtB, cdtC, and cadF were detected in 48.6%, 61.1%, 17.4%, 67.4%, 19.3%, 51%, and 5% of all Campylobacter isolates, respectively. Clonal analysis revealed that isolates along the continuum were highly diverse, with isolates from the same sampling points belonging to the same major ERIC-types. The study showed relatively high resistance to antibiotics commonly used in intensive pig production in South Africa with some evidence, albeit minimal, of transmission across the farm-to-fork continuum. This, together with the virulence profiles present in Campylobacter spp., presents a challenge to food safety and a potential risk to human health, necessitating routine surveillance, antibiotic stewardship, and comprehensive biosecurity in intensive pig production.

scholarly journals Prevalence and Susceptibility Profiles of Methicillin Sensitive Staphylococcus aureus from Community and Hospital Associated Infections

2021 ◽  
Author(s):  
. Preeja ◽  
Sanath Kumar ◽  
Veena A Shetty
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Tertiary Care ◽  
Control Measures ◽  
Patient Treatment ◽  
Diffusion Method ◽  
Care Hospital ◽  
Cross Sectional ◽  
Significant Difference ◽  
Hospital Associated Infections

Introduction: Methicillin Sensitive Staphylococcus aureus(MSSA) is a significant human pathogen, involved in both hospital and community associated settings. MSSA, being more susceptible to antibiotics compared to Methicillin Resistant Staphylococcus aureus(MRSA), is found to acquire Multidrug Resistance (MDR) and with the presence of virulence factors can pose difficulty in patient treatment. Aim: To study the prevalence and antibiotic resistance profile of MSSA from community and hospital associated infections. Materials and Methods: The present cross-sectional study was conducted in tertiary care hospital in Mangalore, Karnataka, India from January 2015 to February 2017. Three hundred and five Staphylococcus aureuswere isolated from various clinical specimens and tested for methicillin susceptibility using cefoxitin disc. Antibiotic resistance profiles against 23 antibiotics were determined by disc diffusion method. The difference was compared for antibiotic sensitivity with respect to Community Associated Methicillin Sensitive Staphylococcus aureus (CA-MSSA) and Hospital Associated Methicillin Sensitive Staphylococcus aureus (HA-MSSA) and Chi-square test was used for statistical analysis. Results: Of 305 MSSA isolated, 219 (71.8%) were CA-MSSA and 86 (28.2%) were HA-MSSA. S. aureus was isolated mostly from Skin and Soft Tissue Infections (SSTI, 61.3%). Resistance was observed against ciprofloxacin (64.6%), erythromycin (43.9%), ofloxacin (42.3%), clindamycin (20.7%), ampicillin (100%) and penicillin (90.5%). There was a significant difference (p<0.05) between the resistance of CA-MSSA and HA-MSSA against cefotaxime and co-trimoxazole. Conclusion: The present study showed the increasing prevalence of MSSA in the community and hospital settings with the emergence of MDR which has to be dealt immediately with appropriate control measures.

Comparison of Cell Sizes of Methicillin-Resistant Staphylococcus aureus with Presence and Absence of the MecA Gene

2021 ◽  
Vol 83 (1) ◽  
pp. 68-77
Author(s):  
O. Berhilevych ◽  
◽  
V. Kasianchuk ◽  
M. Kukhtyn ◽  
P. Shubin ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Cell Size ◽  
Meca Gene ◽  
Morphological Characteristics ◽  
Resistance Mechanisms ◽  
The Body ◽  
Diffusion Method ◽  
Methicillin Resistant ◽  
Wide Range

Staphylococcus aureus is a pathogenic microorganism that causes a wide range of infectious diseases of humans and animals. Staphylococcus aureus produces a large number of toxins, in particular enterotoxins, which enter the body together with food and cause disorders in the gastrointestinal tract. Moreover, S. aureus has several mechanisms of antibiotic resistance, which greatly complicates prevention of bacteria spread as community-acquired and nosocomial infections. The aim of the work was to determine and compare the differences in size of methicillin-resistant strains of S. aureus with different resistance mechanisms by scanning electron microscopy (SEM). Methods. Disc diffusion method was used to establish the mechanism of antibiotic resistance of the obtained isolates. After description of antibiotic resistant and selection of S. aureus isolates with resistance to penicillin and oxicilin, an SEM of the strains and a further comparison of their morphological characteristics, in particular cell size, with the help of Djmaizer v.5.1.10 software was carried out. Results. 54 isolates of S. aureus, obtained from various environmental objects, dairy farms and food products, were tested. PCR revealed sequences of the mecA gene, which is responsible for bacteria resistance to beta-lactams. We determined the cells size of S. aureus isolates resistant to penicillin and oxycillin and performed a comparative analysis of their morphological characteristics using SEM. Conclusions. In the course of the study, it was found that S. aureus isolates with mecA gene (mecA+) have smaller cell size than S. aureus isolates without mecA gene (mecA-).

scholarly journals Prevalence and Antibiotic Resistance Mechanisms of Methicillin-Resistant Staphylococcus aureus isolates from Selected Hospitals in Ashanti Region, Ghana.

2021 ◽  
Author(s):  
Crystal Ngofi Zumbi ◽  
Vivian Etsiapa Boamah ◽  
Yaw Duah Boakye ◽  
Hayford Odoi ◽  
Christian Agyare
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Health Problem ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Efflux Pump ◽  
Meca Gene ◽  
Resistance Mechanisms ◽  
Diffusion Method ◽  
Methicillin Resistant ◽  
Ashanti Region

Abstract Background: Antibiotic resistance in bacteria has long been recognized as a major health problem occurring worldwide. The emergence of methicillin-resistant Staphylococcus aureus (MRSA) remains a global health problem. MRSA is reported as one of the leading pathogens involved in increased rates of morbidity and mortality amongst patients in Ghana. This study determined the prevalence and resistance mechanisms of MRSA isolated from patients in selected hospitals in the Ashanti Region of Ghana. Antibiograms of the isolates were determined using the Kirby-Bauer disk diffusion method. Antibiotic resistance genes (mecA and mecC) were detected and efflux pump activity assessed using polymerase chain reaction (PCR) and microbroth dilution methods, respectively. Results: Out of 626 samples obtained from patients, S. aureus was identified in 68, representing 10.9%. Multidrug resistance (MDR) was observed in 46 (67.6%) of the S. aureus isolates of which 28 (60.9%) were Methicillin Resistant Staphylococcus aureus (MRSA). The MRSA isolates showed higher susceptibility to trimethoprim-sulfamethoxazole (50%) and higher resistance to oxacillin and cefoxitin (100%). mecA gene was identified in 9 (32.1%) of the MRSA isolates whereas mecC gene was absent in all the isolates. The isolates did not exhibit any multiple efflux pump activities. Conclusion: The prevalence of MDR-MRSA in S. aureus infections at healthcare facilities in the Ashanti region was found to be high. The presence of the mecA gene was identified as a possible mechanism responsible for resistance in the MRSA isolates.

scholarly journals From Farm-to-Fork: E. Coli from an Intensive Pig Production System in South Africa Shows High Resistance to Critically Important Antibiotics for Human and Animal Use

Antibiotics ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 178 ◽  
Author(s):  
Shima E. Abdalla ◽  
Akebe Luther King Abia ◽  
Daniel G. Amoako ◽  
Keith Perrett ◽  
Linda A. Bester ◽  
...  
Keyword(s):  
South Africa ◽  
Antibiotic Resistance ◽  
Production System ◽  
Antibiotic Use ◽  
Diffusion Method ◽  
Growth Promoters ◽  
Production Chain ◽  
Pig Production ◽  
E Coli ◽  
Pcr Targeting

Antibiotic resistance profiles of Escherichia coli were investigated in an intensive pig production system in the uMgungundlovu District, South Africa, using the ‘farm-to-fork’ approach. Four hundred seventeen (417) samples were collected from pig and pig products at different points (farm, transport, and abattoir). E. coli was isolated and enumerated using the Colilert® 18/Quanti-Tray® 2000 system. Ten isolates from each Quanti-tray were selected randomly and putatively identified on eosin methylene blue agar. Real-time PCR targeting the uidA gene was used to confirm isolates to the genus level. The Kirby–Bauer disc diffusion method was used to determine the isolates’ antibiotic susceptibility profiles against 20 antibiotics. A total of 1044 confirmed E. coli isolates were obtained across the three critical points in the food chain. Resistance was observed to all the antibiotics tested with the highest and lowest rates obtained against tetracycline (88.5%) and meropenem (0.2%), respectively. Resistance was also observed to chloramphenicol (71.4%), ampicillin (71.1%), trimethoprim-sulfamethoxazole (61.3%), amoxicillin-clavulanate (43.8%), cephalexin (34.3%), azithromycin (23.9%), nalidixic acid (22.1%), cefoxitin (21.1%), ceftriaxone (18.9%), ciprofloxacin (17.3%), cefotaxime (16.9%), gentamicin (15.5%), cefepime (13.8%), ceftazidime (9.8%), amikacin (3.4%), piperacillin-tazobactam (1.2%), tigecycline (0.9%), and imipenem (0.3%). Multidrug resistance (MDR) was observed in 71.2% of the resistant isolates with an overall multiple antibiotic resistance (MAR) index of 0.25, indicating exposure to high antibiotic use environments at the farm level. A high percentage of resistance was observed to growth promoters and antibiotics approved for veterinary medicine in South Africa. Of concern was resistance to critically important antibiotics for animal and human use and the watch and reserve categories of antibiotics. This could have adverse animal and human health consequences from a food safety perspective, necessitating efficient antibiotic stewardship and guidelines to streamline antibiotic use in the food-animal production chain.

scholarly journals Mutation-Based Antibiotic Resistance Mechanism in Methicillin-Resistant Staphylococcus aureus Clinical Isolates

2021 ◽  
Vol 14 (5) ◽  
pp. 420
Author(s):  
Tanveer Ali ◽  
Abdul Basit ◽  
Asad Mustafa Karim ◽  
Jung-Hun Lee ◽  
Jeong-Ho Jeon ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Active Site ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Meca Gene ◽  
Resistance Mechanism ◽  
Ligand Docking ◽  
Clinical Samples ◽  
Allosteric Site ◽  
Methicillin Resistant

β-Lactam antibiotics target penicillin-binding proteins and inhibit the synthesis of peptidoglycan, a crucial step in cell wall biosynthesis. Staphylococcus aureus acquires resistance against β-lactam antibiotics by producing a penicillin-binding protein 2a (PBP2a), encoded by the mecA gene. PBP2a participates in peptidoglycan biosynthesis and exhibits a poor affinity towards β-lactam antibiotics. The current study was performed to determine the diversity and the role of missense mutations of PBP2a in the antibiotic resistance mechanism. The methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical samples were identified using phenotypic and genotypic techniques. The highest frequency (60%, 18 out of 30) of MRSA was observed in wound specimens. Sequence variation analysis of the mecA gene showed four amino acid substitutions (i.e., E239K, E239R, G246E, and E447K). The E239R mutation was found to be novel. The protein-ligand docking results showed that the E239R mutation in the allosteric site of PBP2a induces conformational changes in the active site and, thus, hinders its interaction with cefoxitin. Therefore, the present report indicates that mutation in the allosteric site of PBP2a provides a more closed active site conformation than wide-type PBP2a and then causes the high-level resistance to cefoxitin.

scholarly journals Detection of Methicillin Resistant Staphylococcus aureus in Dairy Products and Anterior Nares of Dairy Workers

2019 ◽  
Vol 6 ◽  
pp. 59-62
Author(s):  
Ranjana K.C. ◽  
Ganga Timilsina ◽  
Anjana Singh ◽  
Supriya Sharma
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Susceptibility ◽  
Dairy Products ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Dairy Product ◽  
Control Measures ◽  
Diffusion Method ◽  
Mannitol Salt Agar ◽  
Methicillin Resistant ◽  
Dairy Workers

Objectives: To isolate methicillin resistant Staphylococcus aureus (MRSA) from anterior nares of dairy workers and dairy products and assess the antibiotic susceptibility pattern of the isolates. Methods: Swab samples collected from anterior nares of dairy workers and dairy product (butter) were inoculated into mannitol salt agar and incubated at 37ºC for 24 hours. Identification was done based on colony characteristics, Gram's staining, catalase, oxidase and coagulase test. Antibiotic susceptibility testing was done by modified Kirby Bauer disc diffusion method. MRSA was confirmed by using cefoxitin disc. Results: A total of 109 S. aureus (98 from dairy workers and 11 from butter samples) were isolated. Out of them 32 MRSA were isolated from dairy workers and 4 from butter samples. The association between age group and MRSA was found insignificant (p = 0.115). The association of MRSA between male and female workers was found significant (>0.05). About 86% of the MRSA isolates were susceptible to Gentamicin (86.11%) followed by Ciprofloxacin (77.78%). Conclusion: Detection of MRSA among dairy workers and dairy products warrants proper handling and adequate control measures to prevent transmission of MRSA from dairy industry.

scholarly journals Prevalence, Antibiogram and Molecular Characterization of Comunity-Acquired Methicillin-Resistant Staphylococcus Aureus in AWKA, Anambra Nigeria

2016 ◽  
Vol 10 (1) ◽  
pp. 211-221 ◽  
Author(s):  
Blessing Ike ◽  
Malachy C. Ugwu ◽  
Moses N. Ikegbunam ◽  
David Nwobodo ◽  
Chika Ejikeugwu ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Binding Protein ◽  
Meca Gene ◽  
Latex Agglutination ◽  
Diffusion Method ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Penicillin Binding Protein ◽  
Carriage Rate ◽  
Molecular Features

Objectives:This study evaluated the prevalence, antibiogram and molecular features of CA-MRSA in Awka, Nigeria.Methods:Confirmation of MRSA was done by testing resistance to oxacillin (1µg), cloxacillin (5µg) and cefoxitin(30µg) on sterile Mueller Hinton agar supplemented with 4% sodium chloride. The MRSA strains were subjected to antimicrobial susceptibility testing using Kirby-Bauer disc diffusion method. Minimum inhibitory concentration was determined using agar dilution method. Penicillin binding protein 2a was detected through rapid latex agglutination assay while mecA gene was detected by polymerase chain reaction. A total of 142S. aureusisolates were obtained from 261 samples sourced from Staff, students and fomites of the Faculty of Pharmaceutical SciencesResult:The overall prevalence of MRSA was 22.6%. The carriage rate was higher in females (56.5%) than male (43.5%) and was highest in individuals of 20-30 years of age (57.65%). The MIC of the oxacillin sodium salt ranged from 4-32 μg/ml. The multi-antibiotic resistance indices show that 53.4% had Multiple Antibiotic Resistance Indexing (MARI) higher than 0.2. Penicillin binding protein 2a was detected in 8.4% of MRSA isolates, all from nasal carriage while mecA gene was detected in 5 of isolates.Conclusion:This study showed a very high prevalence of MRSA carriage among studied subjects.

scholarly journals MULTIPLE ANTIBIOTIC RESISTANCE INDEXING OF NON- FERMENTING GRAM NEGATIVE BACILLI

2017 ◽  
Vol 10 (6) ◽  
pp. 78
Author(s):  
Bhuvaneshwari Gunasekar
Keyword(s):  
Antibiotic Resistance ◽  
Tertiary Care ◽  
Multidrug Resistant ◽  
Diffusion Method ◽  
Care Hospital ◽  
Multiple Antibiotic Resistance ◽  
Antibiotic Resistant ◽  
Gram Negative ◽  
Mar Index ◽  
Surgery Department

Objective: The multiple antibiotic resistance (MAR) indexing and finding Multidrug resistant (MDR) bacteria will help to indicate the origin from high risk of contamination where the antibiotics are often used. Hence this study was carried out to give the MAR index of non-fermenting Gram negative bacilli in a tertiary care hospital which would help our infection control team also.Methods: Drug resistance was tested by Kirby bauer’s disc diffusion method. MAR index was calculated using the formula, a/b (were a= number of antibiotics to which the organism was resistant and b= total number of antibiotics to which the organism was tested).Results: Out of 240 Gram negative non-fermenters isolated, 117 (49%) strains were greater than 0.2 of MAR index, 95(81%) was from in-patient department. 73(62%) were hospitalized for more than 3 days, 44 (38%) was from surgery department. 49(42%) was wound specimen. Out of 117 multiple antibiotic resistant isolates 99 (85%) were MDR isolates.Conclusion: 51% prevalence of isolates >0.2 MAR index shows that the source of contamination can still be brought up down by proper surveillance and management with proper usage of  surface and skin disinfectants especially in surgery ward where the MAR index has indicated more usage of antibiotics

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