Normalized Resistance Interpretation Research Articles

Epidemiological cutoff values and genetic antimicrobial resistance of Lactococcus garvieae and L. petauri

Lactococcus garvieae and L. petauri are etiological agents of lactococcosis, which cause significant losses in trout aquaculture at elevated water temperatures. Optimizing antimicrobial dosage regimens and identifying the minimum inhibitory concentration (MIC) are important prerequisites for determining clinical breakpoints and effective antimicrobial use. In the present study, epidemiological cutoff values (ECV) were established for 14 antimicrobials using L. garvieae (n = 32) and L. petauri (n = 78), isolated from rainbow trout farms in Türkiye, Spain, Italy, and Greece. The provisional ECVs were calculated using the ECOFFinder and normalized resistance interpretation (NRI) approaches. Furthermore, gene cassettes, capsule gene clusters, and the presence of fourteen antibiotic resistance genes (ARGs) that encode resistance against seven antimicrobial agents, were evaluated. The calculated ECVs of antimicrobials ranged from 1.25 μg mL−1 for amoxicillin to 80.25 μg mL−1 for streptomycin. The percentages of wild type (WT) and non-wild type (NWT) isolates differed based on the approach, and ECVs calculated based on ECOFFinder approach were generally higher. Among the ARGs, those encoding resistance against tetracycline (tetA), β-lactamase (blaSHV, blaTEM, and blaOXA), amphenicol (floR), and macrolide (ermA) were detected in <10% of the isolates. The genes encoding resistance against quinolones (gyrA and qnrA) and aminoglycosides (strB) were the most detected genes. While 40.9% of the isolates contained Class 1 integron cassette regions, 33.6% of the isolates contained Class 2 integron cassette regions, and 9.1% of the isolates contained both cassette regions. None of the screened isolates were positive for the capsule gene cluster. Significant but weak correlations were found among and between ARGs and MIC distribution of antibiotics. The results of this study could be useful for identifying WT and non-WT isolates, as well as for susceptibility surveillance.

AMR Threat Perception Assessment of Heterotrophic Bacteria From Shrimp Aquaculture Through Epidemiological Cut off Values.

Emergence and dissemination of antibiotic resistance is one of the major risks associated with the rampant usage of antibiotics in food-producing animals including aquaculture. To determine Epidemiological Cut-OFF (ECOFF) values of heterotrophic bacterial populations from shrimp culture environments against five different antibiotics. In this present study, bacterial samples were isolated from Penaeus vannamei culture environment in different locations of Andhra Pradesh, which is the aquaculture hub of India. The bacterial isolates were assessed for antibiotic resistance towards five antibiotics belonging to different classes (oxytetracycline, chloramphenicol, erythromycin, ciprofloxacin, and co-trimoxazole) by the disc diffusion method. Determination of Epidemiological Cut-OFF (ECOFF) values and analysis by employing normalized resistance interpretation (NRI) was carried out. The most dominant bacterial populations from shrimp culture were Vibrio spp. (pathogenic bacteria) followed by Bacillus spp. (probiotic bacteria). The bacterial isolates showed highest resistance towards oxytetracycline (overall 23.38%) and in location L6 (59.4%) followed by co-trimoxazole (31.1%). ECOFF values calculated by employing NRI showed that the disc diffusion data were distributed in a normalized manner. The maximum ECOFF value was obtained for ciprofloxacin (23.32 mm), while the minimum value was observed for oxytetracycline (9.05 mm). The antibiotic resistant phenotypes showed that the majority of the heterotrophic bacterial isolates (>60%) belonged to the non-wild type phenotype and primarily towards oxytetracycline (90%). The presence of non-wild antibiotic-resistant phenotypes of heterotrophic bacterial populations (which include not only pathogenic bacteria but also probiotic bacteria) indicates that shrimp culture ponds may be a reservoir for drug-resistant bacteria and there is a greater risk associated with transmission of resistant genes across bacterial flora. NRI analysis of antibiotic disc diffusion data of heterotrophic bacterial populations in shrimp aquaculture environments revealed that majority of them belonged to non-wild type (90%) paticularly to oxytetracycline in comparison to other studied antibiotics (chloramphenicol, erythromycin, ciprofloxacin and co-trimoxazole).

Serotype distribution and antibiogram of Streptococcus parauberis isolated from fish in South Korea.

Streptococcus parauberis is the dominant etiological agent of streptococcosis, the most devastating bacterial disease in the olive flounder farming industry in South Korea. In this study, the distribution of serotypes, antimicrobial susceptibility, and presence of antimicrobial resistance genes (ARGs) in S. parauberis isolates obtained between 1999 and 2021 was thoroughly investigated to gain insight into the dynamics of their presence and the relationship between serotypes and antimicrobial resistance. Disk diffusion testing of 103 isolates against 10 antimicrobial agents was performed, and epidemiological cut-off values generated through normalized resistance interpretation analysis were used to classify wild-type (WT) and non-wild-type (NWT) populations. Principal component analysis and hierarchical clustering were implemented to achieve an understanding on the relationship between serotypes and antimicrobial resistance patterns. PCR-based serotyping showed that serotype Ia (67.1%) was the most prevalent in South Korea, followed by serotypes Ib/Ic (25.2%) and II (7.7%). The highest proportion of isolates was assigned to NWT against amoxicillin (80.6%), followed by oxytetracycline (77.7%) and erythromycin (48.5%). The time-scale data showed that recently obtained serotypes Ib/Ic and II isolates tended to be categorized as NWT populations resistant to more antibiotics, possibly due to microbial adaptation to antibiotic pressure. ARGs responsible for resistance to oxytetracycline and erythromycin were found only in NWT populations in serotype Ia [tet(S) and erm(B), respectively], and serotype II [tet(M) and mef(J)-msr(I), respectively]. We also found that the mef-msr gene pair in S. parauberis serotype II might be involved in low-level resistance to erythromycin. IMPORTANCE This study presents serotype distribution and antimicrobial susceptibility data along with the antimicrobial resistance genes (ARGs) of Streptococcus parauberis, which is an important bacterial fish pathogen worldwide. In particular, almost all oxytetracycline and erythromycin non-wild-type (NWT) populations harbored tet(S) or tet(M), and erm(B) or mef(J)-msr(I), respectively. Interestingly, these ARGs were distributed in a highly serotype-dependent manner, resulting in a clear correlation between the antibiogram and serotype distribution. Moreover, recent isolates belonging to serotypes Ib/Ic and II tended to be more frequently categorized as NWT against antimicrobials, including amoxicillin and cefalexin compared to old isolates, while a dramatic decrease in erythromycin and clindamycin NWT frequencies was observed in recent serotype Ia isolates, which lacked erm(B). These variations might be attributed to shifts in the antibiotics employed in South Korean aquaculture over time. The overall findings would provide important background knowledge for understanding the epidemiology of S. parauberis infection in aquaculture.

Comparative analysis of clinical breakpoints, normalized resistance interpretation and epidemiological cut-offs in interpreting antimicrobial resistance of Escherichia coli isolates originating from poultry in different farm types in Tanzania.

Existing breakpoint guidelines are not optimal for interpreting antimicrobial resistance (AMR) data from animal studies and low-income countries, and therefore their utility for analysing such data is limited. There is a need to integrate diverse data sets, such as those from low-income populations and animals, to improve data interpretation. There is very limited research on the relative merits of clinical breakpoints, epidemiological cut-offs (ECOFFs) and normalized resistance interpretation (NRI) breakpoints in interpreting microbiological data, particularly in animal studies and studies from low-income countries. The aim of this study was to compare antimicrobial resistance in Escherichia coli isolates using ECOFFs, CLSI and NRI breakpoints. A total of 59 non-repetitive poultry isolates were selected for investigation based on lactose fermentation on MacConkey agar and subsequent identification and confirmation as E. coli using chromogenic agar and uidA PCR. Kirby Bauer disc diffusion was used for susceptibility testing. For each antimicrobial agent, inhibition zone diameters were measured, and ECOFFs, CLSI and NRI bespoke breakpoints were used for resistance interpretation. According to the interpretation of all breakpoints except ECOFFs, tetracycline resistance was significantly higher (TET) (67.8 -69.5 %), than those for ciprofloxacin (CIPRO) (18.6 -32.2 %), imipenem (IMI) (3.4 -35 %) and ceftazidime (CEF) (1.7 -45.8 %). Prevalence estimates of AMR using CLSI and NRI bespoke breakpoints did not differ for CEF (1.7 % CB and 1.7 % COWT), IMI (3.4 % CB and 4.0 % COWT) and TET (67.8 % CB and 69.5 % COWT). However, with ECOFFs, AMR estimates for CEF, IMI and CIP were significantly higher (45.8, 35.6 and 64.4 %, respectively; P<0.05). Across all the three breakpoints, resistance to ciprofloxacin varied significantly (32.2 % CB, 64.4 % ECOFFs and 18.6 % COWT, P<0.05). AMR interpretation is influenced by the breakpoint used, necessitating further standardization, especially for microbiological breakpoints, in order to harmonize outputs. The AMR ECOFF estimates in the present study were significantly higher compared to CLSI and NRI.

Tentative Epidemiological Cut-Off Values and Distribution of Resistance Genes in Aquatic Pseudomonas Species Isolated from Rainbow Trout

Epidemiological cut-off value (ECV) analysis for commonly used antimicrobials in aquaculture have not been established for many aquatic pathogens, including Pseudomonas. This study was the first to examine the categorization of 92 aquatic Pseudomonas isolates by calculating seven antimicrobials ECVs using two analytical methods: normalized resistance interpretation and ECOFFinder. Pseudomonas spp. isolates had decreased sensitivity to all antimicrobials examined except for doxycycline and ciprofloxacin. The PCR analysis of the 91 isolates of Pseudomonas spp. detected the tetracycline genes are predominant with the count of 41 genes, including tetA, tetC, tetD, tetM, tetS and tetH, following sulfonamide genes are in 21 isolates including sul1 and sul2, floR gene in 15 isolates and ermA gene in three isolates. Our findings provide an understanding of the antimicrobial categorization of Pseudomonas species, which are significant groups, subgroups, and species for aquaculture due to insufficiently defined breakpoints or cut-off values reported in CLSI and/or EUCAST.

Setting of the tentative epidemiological cut-off values of contezolid for Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae and Streptococcus agalactiae.

To set the tentative epidemiological cut-off values (TECOFFs) of contezolid for Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae and Streptococcus agalactiae based on the distributions of inhibition zone diameters and MICs. A total of 1358 non-duplicate clinical isolates of Gram-positive bacteria were collected from the patients across China from 2017 to 2020. The isolates were tested for susceptibility to contezolid and the comparator linezolid by broth microdilution and disc diffusion methods in three microbiology laboratories. The zone diameters and MICs of linezolid WT strains were used to set the WT TECOFFs of contezolid by normalized resistance interpretation calculations. Contezolid showed an aggregate MIC range from 0.03 to 8 mg/L and MIC90 value of 1-2 mg/L against all of the Gram-positive bacterial strains tested. The TECOFF of contezolid based on MIC distributions was 4 mg/L for both S. aureus and Enterococcus species, and 2 mg/L for S. pneumoniae and S. agalactiae. The TECOFF of contezolid based on zone diameter was 24 mm for S. aureus, 18 mm for E. faecalis, 20 mm for E. faecium and S. pneumoniae, and 17 mm for S. agalactiae. The epidemiological cut-off values of contezolid were set tentatively for selected Gram-positive bacteria using the MIC and zone diameter distributions. These data are helpful for clinical microbiologists and clinicians to interpret the antimicrobial susceptibility results of contezolid.

Epidemiological Cut-Off Values and Multidrug Resistance of Escherichia coli Isolated from Domesticated Poultry and Pigs Reared in Mwanza, Tanzania: A Cross-Section Study.

Simple SummaryThe objectives of this study were to determine the prevalence of multidrug resistance phenotypes and the distribution of Escherichia coli among poultry and pigs. Laboratory procedures were conducted according to standard operating procedures and international guidelines. Our findings showed that poultry and pigs reared in Mwanza, Tanzania, are colonized with resistant bacterial phenotypes. Further, different populations of intestinal flora, E. coli, exist between poultry and pigs.Increasing antimicrobial resistance (AMR) colonizing domesticated animals is a global concern threatening food safety. This study aimed at determining the prevalence of multidrug resistance (MDR) and epidemiological cut-off values (ECVs) of E. coli isolated from poultry and pigs in Mwanza, Tanzania. This cross-sectional study was conducted between June and August 2021, involving 297 pigs, 191 broilers, and 203 layers. Rectal and cloacal swabs were collected and processed following standard guidelines. ECVs were determined using normalized resistance interpretation (NRI), a computer software, and descriptive analysis was performed using STATA version 13.0. The overall prevalence of MDR E. coli was 63.2%, whereas poultry (87.5% layers and 86.3% broilers) were more colonized than pigs (31.8%) (p < 0.001). Based on ECVs of antibiotics tested, E. coli from broilers, layers, and pigs exhibited different resistance patterns hence different populations. Exotic breed (p < 0.001) and recent antimicrobial use (p < 0.001) significantly predicted colonization with MDR E. coli. Veterinary officers should implement regulations that prohibit the inappropriate use of antimicrobial agents in livestock keeping.

Evaluation of the in vitro susceptibility of Tenacibaculum dicentrarchi to tiamulin using minimum inhibitory concentration tests.

Tenacibaculosis caused by Tenacibaculum dicentrarchi is the third most important bacterial fish infection affecting the Chilean salmon industry. Losses to this disease are most frequently controlled by treatments with florfenicol and oxytetracycline. However, recent tenacibaculosis outbreaks were controlled through the extra-label, oral administration of tiamulin, resulting in high treatment efficiency. In this study, we present an analysis of susceptibility patterns of 32T.dicentrarchi isolates and the type strain CECT 7612T to tiamulin by determining the minimum inhibitory concentrations (MICs) according to the procedures recommended by the Clinical and Laboratory Standard Institute, but fixing incubation temperature to the more appropriate for the growth of T.dicentrarchi (18 ºC). The MICs of the T.dicentrarchi isolates were unimodally distributed (0.06-1.0µg/ml range), while the CECT 7612T strain presented an MIC of 0.5μg/ml. Calculations using Normalized Resistance Interpretation provided epidemiological cut-off values of ≤1.0µg/ml, with the 33T.dicentrarchi classified as wild type. In Chile, tiamulin is authorized for use in other livestock species, but application in salmonids is extra-label. Our presented in vitro results suggest that tiamulin is a viable alternative to florfenicol, specifically as tiamulin requires comparatively lower concentrations to inhibit T.dicentrarchi. Considering that tiamulin is also exclusively for veterinary use, is classified as "least important" by the World Health Organization and has not resulted in the development of bacterial resistance, pharmaceutical companies should be requested to register tiamulin and provide alternative antimicrobial treatments for the salmonid industry.

Establishment of Epidemiological Cut-Off Values and the Distribution of Resistance Genes in Aeromonas hydrophila and Aeromonas veronii Isolated from Aquatic Animals.

The emergence of antimicrobial-resistant bacteria is an enormous challenge to public health. Aeromonas hydrophila and Aeromonas veronii are opportunistic pathogens in fish. They exert tremendous adverse effects on aquaculture production, owing to their acquired antibiotic resistance. A few Clinical and Laboratory Standards Institute (CLSI) epidemiological cut-off values (ECVs) against Aeromonas spp. are available. We evaluated antimicrobial susceptibility by establishing 8 ECVs using two analytical methods, normalized resistance interpretation and ECOFFinder. We detected antimicrobial resistance genes in two motile Aeromonas spp. isolated from aquatic animals. Results showed that 89.2% of A. hydrophila and 75.8% of A. veronii isolates were non-wild types according to the oxytetracycline ECVCLSI and ECVNRI, respectively. The antimicrobial resistance genes included tetA, tetB, tetD, tetE, cat, floR, qnrA, qnrB, qnrS, strA-strB, and aac(6′)-1b. The most common tet gene in Aeromonas spp. isolates was tetE, followed by tetA. Some strains carried more than one tet gene, with tetA–tetD and tetA–tetE found in A. hydrophila; however, tetB was not detected in any of the strains. Furthermore, 18.6% of A. hydrophila and 24.2% of A. veronii isolates showed presumptive multidrug-resistant phenotypes. The emergence of multidrug resistance among aquatic aeromonads suggests the spread of drug resistance and difficult to treat bacterial infections.

Local epidemiological cutoff values and antimicrobial susceptibility profile for Brazilian Francisella orientalis isolates

Francisellosis is an emerging disease caused by Francisella orientalis and is responsible for major economic losses in tilapia farms worldwide. The method of choice for francisellosis outbreak control is oral antibiotic administration, as no commercial vaccines or autogenous effective vaccines are available. Although there is a standard method recommended by the Clinical and Laboratory Standards Institute (CLSI) for susceptibility testing of fish pathogenic Francisella spp., basic information on culture and growth conditions for this pathogen is scarce. In this study, the antibiotic susceptibility profile of Francisella orientalis was determined using the disk diffusion test. For that, 89 Francisella orientalis samples obtained from disease outbreaks in tilapia farms in 2012–2019 in various Brazilian states were selected for testing against different antibiotics. Because F. orientalis cannot grow on the standard Mueller-Hinton (MH) agar medium, two different culture media were selected for testing: modified heart cystine agar (CHAH) and modified cation-adjusted Mueller-Hinton agar (MMHA). Different incubation periods were also tested for more appropriate and accurate measurement data. CHAH was more suitable for performing the diffusion disk tests because MMHA required longer incubation periods and formed overestimated inhibition zones. Incubation time affected the antibiogram results mainly for β-lactam antibiotics, with 48 h being the ideal period for measuring inhibition zones. Epidemiological cutoff values of Francisella orientalis isolates using the normalized resistance interpretation (NRI) methodology were successfully calculated. All 89 isolates (100%) were considered wild type (WT) for florfenicol, oxytetracycline, erythromycin, and doxycycline, whereas 86 isolates (96.6%) were considered WT for amoxicillin, ampicillin, enrofloxacin, and norfloxacin. Three isolates (3.4%) were considered non-wild type (NWT) for enrofloxacin, norfloxacin, amoxicillin, and ampicillin. This is the first report of Francisella orientalis isolates resistant to amoxicillin and fluoroquinolones. This study provided important data on Francisella orientalis antibiotic susceptibility. Moreover, it showed that Brazil is already suffering the effects of the inadvertent use of licensed drugs and of off-label drugs, as the aforementioned drug classes are illegally used for farmed fish in the country.

Pathogenicity of Shiga toxin-producing Escherichia coli (STEC) from wildlife: Should we care?

Shiga toxin-producing Escherichia coli (STEC) is one of the most frequent bacterial agents associated with food-borne outbreaks in Europe. In humans, the infection can lead to life-threatening diseases. Domestic and wild animals can harbor STEC, and ruminants are the main STEC reservoirs, although asymptomatic. In the present study we have characterized STEC from wildlife (wild boar (n = 56), red deer (n = 101), red fox (n = 37) and otter (n = 92)). Cultivable STEC (n = 52) were isolated from 17% (n = 49) of the faecal samples. All the isolates were non-O157 STEC encoding stx1 (n = 2; 4%) and/or stx2 genes (n = 51; 98%). Only one strain (2%) isolated from red fox had an antibiotic resistant phenotype. However, when the normalized resistance interpretation of epidemiological cutoffs (NRI ECOFFs) were used, 23% (n = 12) of the strains were non-wildtype to at least one of the antibiotics tested. After analysis by pulsed-field gel electrophoresis (PFGE), 20 strains were selected for whole genome sequencing and belonged to the following serotypes: O27:H30 (n = 15), O146:H28 (n = 2), O146:H21 (n = 1), O178:H19 (n = 1), and O103:H2 (n = 1). In addition to stx, all strains encode several virulence factors such as toxins, adhesins, fimbriae and secretion systems, among others. All sequenced genomes carried several mobile genetic elements (MGEs), such as prophages and/or plasmids. The core genome and the phylogenetic analysis showed close evolutionary relationships between some of the STEC recovered from wildlife and strains of clinical origin, highlighting their pathogenic potential. Overall, our results show the zoonotic potential of STEC strains originating from wildlife, highlighting the importance of monitoring their genomic characteristics following a One Health perspective, in which the health of humans is related to the health of animals, and the environment.

Proposal of Epidemiological Cutoff Values for Apramycin 15 μg and Florfenicol 30 μg Disks Applicable to Staphylococcus aureus.

Apramycin and florfenicol are two antimicrobial agents exclusively used in veterinary medicine. Resistance determinants to these antimicrobial agents have been described in several staphylococci, yet no inhibition zone-based epidemiological cutoff (ECOFF) values are available to detect populations harboring resistance mechanisms. In this study, we propose disk diffusion inhibition zone ECOFF values of Staphylococcus aureus for apramycin and florfenicol. The susceptibility to apramycin and florfenicol was evaluated by disk diffusion of five S. aureus collections, comprising 352 isolates of animal (n = 265) and human (n = 87) origin. The aggregated distributions of inhibition zone diameters were analyzed by the normalized resistance interpretation method to obtain normalized wild-type (WT) population distributions and corresponding ECOFF values. The putative WT populations of S. aureus were characterized by an inhibition zone ≥15 mm (ECOFF = 15 mm) for apramycin and ≥21 mm for florfenicol (ECOFF = 21 mm). Five nonwild-type (NWT) isolates were detected for apramycin, all without inhibition zone and harboring the apmA gene, whereas five NWT isolates were identified for florfenicol, all carrying the fexA gene. The proposed ECOFF values for apramycin and florfenicol may be a valuable tool in future antimicrobial resistance monitoring and surveillance studies to identify S. aureus NWT populations toward these antimicrobial agents.

Comparison of Phenotypical Antimicrobial Resistance between Clinical and Non-Clinical E. coli Isolates from Broilers, Turkeys and Calves in Four European Countries.

Livestock data on antimicrobial resistance (AMR) are commonly collected from bacterial populations of clinical and non-clinical isolates. In contrast to data on non-clinical isolates from livestock, data on clinical isolates are not harmonized in Europe. The Normalized Resistance Interpretation (NRI) method was applied to overcome the lack of harmonization of laboratory methods and interpretation rules between monitoring systems. Statistical analyses were performed to identify associations between the isolate type (clinical vs. non-clinical) and resistance to four antimicrobials (ampicillin, tetracycline, gentamicin, and nalidixic acid) per animal category in Germany and France. Additional statistical analyses comparing clinical and non-clinical isolates were performed with the available data on the same antimicrobial panel and animal categories from the UK and Norway. Higher resistance prevalence was found in clinical isolates compared to non-clinical isolates from calves to all antimicrobials included in Germany and France. It was also found for gentamicin in broilers from France. In contrast, in broilers and turkeys from Germany and France and in broilers from the UK, a higher resistance level to ampicillin and tetracycline in non-clinical isolates was encountered. This was also found in resistance to gentamicin in isolates from turkeys in Germany. Resistance differed within countries and across years, which was partially in line with differences in antimicrobial use patterns. Differences in AMR between clinical and non-clinical isolates of Escherichia coli are associated with animal category (broiler, calf, and turkey) and specific antimicrobials. The NRI method allowed comparing results of non-harmonized AMR systems and might be useful until international harmonization is achieved.

Research Note: Epidemiological cutoff values and acquired resistance mechanisms of three veterinary antibiotics against Escherichia coli from chicken respiratory tract infections

Florfenicol, apramycin, and danofloxacin are antibiotics approved only for veterinary use and that have good therapeutic effects on chicken respiratory infections caused by Escherichia coli. We established epidemiological cutoff values (ECV) for these antibiotics using 363 E. coli isolates from tracheal samples of chickens in 5 veterinary clinics in Guangdong Province, China. The minimum inhibitory concentrations (MIC) were determined using the agar dilution method as per Clinical and Laboratory Standards Institution guidelines. The ECV were then calculated using the statistical method and verified by normalized resistance interpretation and ECOFFinder software programs. The ECV of florfenicol, apramycin, and danofloxacin against E. coli were 16, 16, and 0.125 μg/mL, respectively. Susceptibility tests indicated that these isolates were resistant to florfenicol (66.7%), apramycin (22.3%), and danofloxacin (92.3%). Strains carrying floR were distributed in the range of MIC ≥32 μg/mL for florfenicol. Apramycin resistance was found in 77 strains (77/363, 21.1%), and isolates that carried aac(3)-IV were all in the range of MIC ≥512 μg/mL. Danofloxacin resistance was found in the range of MIC ≤0.125 μg/mL, but there were no mutations in the quinolone resistance–determining regions and plasmid-mediated quinolone resistance genes qnrA, qnrB, qnrC, qnrD, aac-(6′)-Ib-cr, qep, and oqxB. The presence of the qnrS gene was verified in a few of the strains with an MIC of 0.06 μg/mL. The establishment of ECV was significant for monitoring of resistance development and therapy guidance.

Antimicrobial resistance in ovine bacteria: A sheep in wolf's clothing?

BackgroundTo monitor the prevalence of antimicrobial resistance (AMR), methods for interpretation of susceptibility phenotypes of bacteria are needed. Reference limits to declare resistance are generally based on or dominated by data from human bacterial isolates and may not reflect clinical relevance or wild type (WT) populations in livestock or other hosts.MethodsWe compared the observed prevalence of AMR using standard and bespoke interpretations based on clinical breakpoints or epidemiological cut-offs (ECOFF) using gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria from sheep as exemplars. Isolates were obtained from a cross-sectional study in three lowland sheep flocks in Scotland, and from a longitudinal study in one flock in Norway. S. aureus (n = 101) was predominantly isolated from milk or mammary glands whilst E. coli (n = 103) was mostly isolated from faecal samples. Disc diffusion testing was used to determine inhibition zone diameters, which were interpreted using either clinical breakpoints or ECOFF, which distinguish the bacterial wild type population from bacteria with acquired or mutational resistance to the compound of interest (non-wild type). Standard ECOFF values were considered as well as sheep-specific values calculated from the data using Normalized Resistance Interpretation (NRI) methodology.ResultsThe prevalence of AMR as measured based on clinical breakpoints was low, e.g. 4.0% for penicillin resistance in S. aureus. Estimation of AMR prevalence based on standard ECOFFs was hampered by lack of relevant reference values. In addition, standard ECOFFS, which are predominantly based on human data, bisected the normal distribution of inhibition zone diameters for several compounds in our analysis of sheep isolates. This contravenes recommendations for ECOFF setting based on NRI methodology and may lead to high apparent AMR prevalence. Using bespoke ECOFF values based on NRI, S. aureus showed non-wild type for less than 4% of isolates across 13 compounds, and ca. 13% non-wild type for amoxicillin and ampicillin, while E. coli showed non-wild type for less than 3% of isolates across 12 compounds, and ca. 13% non-wild type for tetracyclines and sulfamethoxazole-trimethoprim.ConclusionThe apparent prevalence of AMR in bacteria isolated from sheep is highly dependent on interpretation criteria. The sheep industry may want to establish bespoke cut-off values for AMR monitoring to avoid the use of cut-offs developed for other host species. The latter could lead to high apparent prevalence of resistance, including to critically important antimicrobial classes such as 4th generation cephalosporins and carbapenems, suggesting an AMR problem that may not actually exist.

The determination of antimicrobial susceptibility by MIC and epidemiological cut-off values and the detection of resistance genes in Aeromonas species isolated from cultured fish.

The present study was aimed at determining antimicrobial susceptibility by a CLSI standard microdilution testing protocol and detecting the resistance genes of motile Aeromonas species isolated from cultured fish. The importance of the minimum inhibitory concentrations was assessed based on statistically determined epidemiological cut-off values calculated by normalized resistance analysis. Unfortunately, CLSI epidemiological cut-off values are available only for Aeromonas salmonicida, and there is no further detailed data on Aeromonas isolated from aquatic animals. The antimicrobial susceptibilities of pre-identified motile Aeromonas species to florfenicol, tetracycline and sulfamethoxazole were determined by calculating epidemiological cut-off values with fully automated and freely available Excel spreadsheets, applying the normalized resistance interpretation (NRI) method. Furthermore, the presence of the antimicrobial resistance genes floR, tetA, tetB, tetC, tetD, tetE, tetH, sulI, sulII and sulIII was detected by PCR analysis and confirmed by sequence analysis. The presence of up to six different genes (multiple antimicrobial resistance) was determined in the Aeromonas isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: In this study, we investigated phenotypic and genotypic antimicrobial resistance characteristics by a novel method based on epidemiological cut-off values. This is the second comprehensive study on the antimicrobial susceptibility characteristics of Aeromonas species using NRI and epidemiological cut-off values. The present research is related to our previous researches focussed on the identification of motile Aeromonads, their prevalence in relation to different fish lengths, seasons and regions, and covered the investigation of Lactococcus garvieae, Yersinia ruckeri, Flavobacterium spp., Enterobacter spp. and Citrobacter spp.

Assessment of the Usefulness of Cefapirin and Cefalonium Disks for Susceptibility Testing of Staphylococcus aureus Isolates from Bovine Mastitis.

Cefapirin (CEP) and cefalonium (CNM) are first-generation cephalosporins widely used to treat bovine mastitis caused by Gram-positive bacteria including staphylococci. However, disks for susceptibility testing of those drugs in causative bacteria are not available. This study evaluated the efficacy of 10 µg and 30 µg pilot disks of CEP (CEP10 and CEP30) and CNM (CNM10 and CNM30) against 130 Staphylococcus aureus isolates from bovine mastitis. Scattergrams of minimum inhibitory concentrations (MICs) and zone diameters (ZDs) illustrated significant correlations between the MICs and ZDs of CEP10 (r = −0.912), CEP30 (r = −0.933), CNM10 (r = −0.847), and CNM30 (r = −0.807). The analysis by Normalized Resistance Interpretation indicated that the epidemiolocal cut-off value (ECV) of MIC for both cefapirin and cefalonium is ≤ 0.5 µg/mL, and the ECV of ZD for CEP10, CEP30, CNM10, and CNM30 were ≥ 22 mm, ≥ 25 mm, ≥ 22 mm, and ≥ 29 mm, respectively. We believe that both 10 μg and 30 μg CEP and CNM susceptibility disks will be helpful for guiding the appropriate use of these antibiotics for bovine mastitis. Further studies toward the establishment of clinical breakpoint of CEP and CNM would be needed for their routine use.

Optimization of florfenicol dose against Piscirickettsia salmonis in Salmo salar through PK/PD studies.

Salmonid Rickettsial Septicemia (SRS) is the disease of greatest economic importance in the Chilean salmon farming industry, causing high mortality in fish during the final stage of their productive cycle at sea. Since current, commercially available vaccines have not demonstrated the expected efficacy levels, antimicrobials, most commonly florfenicol, are still the main resource for the treatment and control of this pathogen. The aim of this study was to determine the most appropriate single dose of florfenicol, administered through medicated feed, for the treatment of Piscirickettsia salmonis (P. salmonis), using pharmacokinetic/pharmacodynamic (PK/PD) models. Previously, Minimum Inhibitory Concentrations (MICs) of florfenicol were determined for 87 P. salmonis isolates in order to define the epidemiological cut-off point (COWT). The most commonly observed MIC was 0.125 μg mL-1 (83.7%). The COWT value was 0.25 μg mL-1 with a standard deviation of 0.47 log2 μg mL-1 and 0.36 log2 μg mL-1, for Normalized resistance interpretation (NRI) method and ECOFFinder method, respectively. A MIC of 1 μg mL-1 was considered the pharmacodynamic value (PD) to define PK/PD indices. Three doses of florfenicol were evaluated in fish farmed under controlled conditions. For each dose, 150 fish were used and blood plasma samples were collected at different time points (0–48 hours). PK parameters were obtained from curves representing plasma concentrations as a function of time. The results of Monte Carlo simulation indicate that at a dose of 20 mg/Kg l.w. of florfenicol, administered orally as medicated feed, there is 100% probability (PTA) of achieving the desired efficacy (AUC0-24h/MIC>125). According to these results, we suggest that at the indicated dose, the PK/PD cut-off point for florfenicol versus P. salmonis could be 2 μg mL-1 (PTA = 99%). In order to assess the indicated dose in Atlantic salmon, fish were inoculated with P. salmonis LF-89 strain and then treated with the optimized dose of florfenicol, 20 mg/Kg bw for 15 days.

Posaconazole MIC Distributions for Aspergillus fumigatus Species Complex by Four Methods: Impact of cyp51A Mutations on Estimation of Epidemiological Cutoff Values.

Estimating epidemiological cutoff endpoints (ECVs/ECOFFS) may be hindered by the overlap of MICs for mutant and nonmutant strains (strains harboring or not harboring mutations, respectively). Posaconazole MIC distributions for the Aspergillus fumigatus species complex were collected from 26 laboratories (in Australia, Canada, Europe, India, South and North America, and Taiwan) and published studies. Distributions that fulfilled CLSI criteria were pooled and ECVs were estimated. The sensitivity of three ECV analytical techniques (the ECOFFinder, normalized resistance interpretation [NRI], derivatization methods) to the inclusion of MICs for mutants was examined for three susceptibility testing methods (the CLSI, EUCAST, and Etest methods). The totals of posaconazole MICs for nonmutant isolates (isolates with no known cyp51A mutations) and mutant A. fumigatus isolates were as follows: by the CLSI method, 2,223 and 274, respectively; by the EUCAST method, 556 and 52, respectively; and by Etest, 1,365 and 29, respectively. MICs for 381 isolates with unknown mutational status were also evaluated with the Sensititre YeastOne system (SYO). We observed an overlap in posaconazole MICs among nonmutants and cyp51A mutants. At the commonly chosen percentage of the modeled wild-type population (97.5%), almost all ECVs remained the same when the MICs for nonmutant and mutant distributions were merged: ECOFFinder ECVs, 0.5 μg/ml for the CLSI method and 0.25 μg/ml for the EUCAST method and Etest; NRI ECVs, 0.5 μg/ml for all three methods. However, the ECOFFinder ECV for 95% of the nonmutant population by the CLSI method was 0.25 μg/ml. The tentative ECOFFinder ECV with SYO was 0.06 μg/ml (data from 3/8 laboratories). Derivatization ECVs with or without mutant inclusion were either 0.25 μg/ml (CLSI, EUCAST, Etest) or 0.06 μg/ml (SYO). It appears that ECV analytical techniques may not be vulnerable to overlap between presumptive wild-type isolates and cyp51A mutants when up to 11.6% of the estimated wild-type population includes mutants.

Antimicrobial susceptibility of Flavobacterium psychrophilum isolates from the United Kingdom.

Routine application of antimicrobials is the current treatment of choice for rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum. In this study, the antimicrobial susceptibilities of 133 F.psychrophilum isolates, 118 of which were from the UK, were evaluated by broth microdilution and disc diffusion methods following VET04-A2 and VET03-A guidelines of Clinical and Laboratory Standards Institute (CLSI), respectively. Isolates were categorized as wild type (fully susceptible, WT) or non-wild type (NWT) using normalized resistance interpretation (NRI)-determined cut-off values (COWT ). Broth microdilution testing showed that only 12% of UK isolates were WT to oxolinic acid (MIC COWT ≤0.25mg/L) and 42% were WT for oxytetracycline (MIC COWT ≤0.25mg/L). In contrast, all the isolates tested were WT (MIC COWT ≤2mg/L) for florfenicol, the main antimicrobial for RTFS control in the UK. Disc diffusion-based COWT values were ≥51mm for 10μg amoxicillin, ≥44mm for 30μg florfenicol, ≥30mm for 2μg oxolinic acid and ≥51mm for 30μg oxytetracycline. There was a high categorical agreement between the classifications of the isolates by two testing methods for florfenicol (100%), oxytetracycline (93%) and oxolinic acid (99%).