Genotypic Antimicrobial Resistance Research Articles

High-Throughput Detection and Characterization of Antimicrobial Resistant Enterococcus sp. Isolates from GI Tracts of European Starlings Visiting Concentrated Animal Feeding Operations.

Antimicrobial resistant enteric bacteria can easily contaminate the environment and other vehicles through the deposition of human and animal feces. In turn, humans can be exposed to these antimicrobial resistant (AMR) bacteria through contaminated food products and/or contaminated drinking water. As wildlife are firmly established as reservoirs of AMR bacteria and serve as potential vectors in the constant spread of AMR, limiting contact between wildlife and livestock and effective tracking of AMR bacteria can help minimize AMR dissemination to humans through contaminated food and water. Enterococcus spp., which are known opportunistic pathogens, constantly found in gastrointestinal tracts of mammalian and avian species, swiftly evolve and cultivate AMR genotypes and phenotypes, which they easily distribute to other bacteria, including several major bacterial pathogens. In this study, we evaluated the use of high throughput detection and characterization of enterococci from wildlife [European starlings (Sturnus vulgaris)] by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) following culture-based isolation. MALDI-TOF MS successfully identified 658 Enterococcus spp. isolates out of 718 presumptive isolates collected from gastrointestinal tracts of European starlings, which were captured near livestock operations in Colorado, Iowa, Kansas, Missouri, and Texas; antimicrobial susceptibility testing was then performed using 13 clinically significant antibiotics.

Genomic analyses of multidrug-resistant Salmonella Indiana, Typhimurium, and Enteritidis isolates using MinION and MiSeq sequencing technologies.

We sequenced 25 isolates of phenotypically multidrug-resistant Salmonella Indiana (n = 11), Typhimurium (n = 8), and Enteritidis (n = 6) using both MinION long-read [SQK-LSK109 and flow cell (R9.4.1)] and MiSeq short-read (Nextera XT and MiSeq Reagent Kit v2) sequencing technologies to determine the advantages of each approach in terms of the characteristics of genome structure, antimicrobial resistance (AMR), virulence potential, whole-genome phylogeny, and pan-genome. The MinION reads were base-called in real-time using MinKnow 3.4.8 integrated with Guppy 3.0.7. The long-read-only assembly, Illumina-only assembly, and hybrid assembly pipelines of Unicycler 0.4.8 were used to generate the MinION, MiSeq, and hybrid assemblies, respectively. The MinION assemblies were highly contiguous compared to the MiSeq assemblies but lacked accuracy, a deficiency that was mitigated by adding the MiSeq short reads through the Unicycler hybrid assembly which corrected erroneous single nucleotide polymorphisms (SNPs). The MinION assemblies provided similar predictions of AMR and virulence potential compared to the MiSeq and hybrid assemblies, although they produced more total false negatives of AMR genotypes, primarily due to failure in identifying tetracycline resistance genes in 11 of the 19 MinION assemblies of tetracycline-resistant isolates. The MinION assemblies displayed a large genetic distance from their corresponding MiSeq and hybrid assemblies on the whole-genome phylogenetic tree, indicating that the lower read accuracy of MinION sequencing caused incorrect clustering. The pan-genome of the MinION assemblies contained significantly more accessory genes and less core genes compared to the MiSeq and hybrid assemblies, suggesting that although these assemblies were more contiguous, their sequencing errors reduced accurate genome annotations. Our research demonstrates that MinION sequencing by itself provides an efficient assessment of the genome structure, antimicrobial resistance, and virulence potential of Salmonella; however, it is not sufficient for whole-genome phylogenetic and pan-genome analyses. MinION in combination with MiSeq facilitated the most accurate genomic analyses.

Genetic capitalism and stabilizing selection of antimicrobial resistance genotypes in Escherichia coli.

Antimicrobial resistance (AMR) in pathogenic strains of bacteria, such as Escherichia coli (E.coli), adversely impacts personal and public health. In this study, we examine competing hypotheses for the evolution of AMR including (i) 'genetic capitalism' in which genotypes that confer antibiotic resistance are gained and not often lost in lineages, and (ii) 'stabilizing selection' in which genotypes that confer antibiotic resistance are gained and lost often. To test these hypotheses, we assembled a dataset that includes annotations for 409 AMR genotypes and a phylogenetic tree based on genome-wide single nucleotide polymorphisms from 29 255 isolates of E.coli collected over the past 134years. We used phylogenetic methods to count the times each AMR genotype was gained and lost across the tree and used model-based clustering of the genotypes with respect to their gain and loss rates. We demonstrate that many genotypes cluster to support the hypothesis for genetic capitalism while a few genotypes cluster to support the hypothesis for stabilizing selection. Comparing the sets of genotypes that fall under each of the hypotheses, we found a statistically significant difference in the breakdown of resistance mechanisms through which the AMR genotypes function. The result that many AMR genotypes cluster under genetic capitalism reflects that strong positive selective forces, primarily induced by human industrialization of antibiotics, outweigh the potential fitness costs to the bacterial lineages for carrying the AMR genotypes. We expect genetic capitalism to further drive bacterial lineages to resist antibiotics. We find that antibiotics that function via replacement and efflux tend to behave under stabilizing selection and thus may be valuable in an antibiotic cycling strategy.

Extensively-Drug Resistant Klebsiella pneumoniae Recovered From Neonatal Sepsis Cases From a Major NICU in Egypt.

BackgroundNeonatal sepsis is a nuisance to clinicians and medical microbiologists, particularly those cases caused by Klebsiella pneumoniae. Thus, we aimed at investigating the profile and mechanisms of antibiotic resistance and the clonal relationships between K. pneumoniae isolated from neonates at the largest tertiary care hospital’s neonatal intensive care units (NICUs) in Minia, Egypt.MethodsThis study comprised 156 neonates diagnosed with culture-proven sepsis from February 2019 to September 2019, at a major NICU of Minia City. All K. pneumoniae isolates were collected and characterized by antimicrobial profile, resistance genotype, and pulsed-field gel electrophoresis typing.ResultsTwenty-four K. pneumoniae isolates (15.3%) were collected out of the 156 sepsis diagnosed neonates. These samples showed extensive drug resistance (XDR) to most of the tested antimicrobials, except fluoroquinolones. All the K. pneumoniae isolates possessed blaVIM and blaNDM carbapenemase genes, while blaKPC gene was detected in 95.8%. Considering extended-spectrum β-lactamases genes, blaCTX–M was found in all the isolates and blaOXA–1 gene in 75% of them. The plasmid-mediated quinolone resistance gene qnrS, was predominantly found among our isolates in comparison to qnrB or qnrA. A moderate degree of clonal relatedness was observed between the isolates.ConclusionTo the best of our knowledge, this the first report of an alarming occurrence of XDR among K. penumoniae isolates recovered from neonatal sepsis in Egypt. Our data necessitate proper antimicrobial stewardship as the choices will be very limited.

Novel Quinolone Nonsusceptible Streptococcus canis Strains with Point Mutations in Quinolone Resistance-Determining Regions and Their Related Factors.

This study investigated quinolone nonsusceptible Streptococcus canis with point mutations in quinolone resistance-determining regions (QRDRs). After selecting targets from 185 isolates, we tested antimicrobial susceptibility using levofloxacin, ciprofloxacin, norfloxacin, and moxifloxacin. We also determined the amino acid sequences of QRDRs in gyrA/gyrB/parC/parE genes and their point mutations. Finally, we performed S. canis-derived M-like protein (SCM) allele typing, multilocus sequence typing, and antimicrobial resistance genotyping. Correlations between nonsusceptible strains and their related factors were examined. We found 13 (7.0%) nonsusceptible isolates consisting of two classes, high-level minimum inhibitory concentrations (MICs) (n = 7, 3.8%) and low-level MICs (n = 6, 3.2%). Mutations Ser81Phe/Ser81Tyr/Glu85Lys in gyrA, Ser67Phe/Ser67Tyr/Asp71Tyr in parC, Asp438Asn in parE, and Gly408Asp in gyrB were observed in these nonsusceptible strains. Common mutations included Ser81 and Ser67/Asp71; additionally, we found one strain each with Glu85, Asp438, and Gly408 mutations. There was a significant correlation between nonsusceptible isolates and the presence of SCM allele type 2, sequence type 46, tetracyclineresistance genes, and macrolide/lincosamide-resistance genes. These results could be used in future, by veterinarians while treating companion animals with clinical symptoms of streptococcal infections.

The Role of European Starlings (Sturnus vulgaris) in the Dissemination of Multidrug-Resistant Escherichia coli among Concentrated Animal Feeding Operations

Antimicrobial use in livestock production is a driver for the development and proliferation of antimicrobial resistance (AMR). Wildlife interactions with livestock, acquiring associated AMR bacteria and genes, and wildlife’s subsequent dispersal across the landscape are hypothesized to play an important role in the ecology of AMR. Here, we examined priority AMR phenotypes and genotypes of Escherichia coli isolated from the gastrointestinal tracts of European starlings (Sturnus vulgaris) found on concentrated animal feeding operations (CAFOs). European starlings may be present in high numbers on CAFOs (>100,000 birds), interact with urban environments, and can migrate distances exceeding 1,500 km in North America. In this study, 1,477 European starlings from 31 feedlots in five U.S. states were sampled for E. coli resistant to third generation cephalosporins (3G-C) and fluoroquinolones. The prevalence of 3G-C and fluoroquinolone-resistant E. coli was 4% and 10%, respectively. Multidrug resistance in the E. coli isolates collected (n = 236) was common, with the majority of isolates displaying resistance to six or more classes of antibiotics. Genetic analyses of a subset of these isolates identified 94 genes putatively contributing to AMR, including seven class A and C β-lactamases as well as mutations in gyrA and parC recognized to confer resistance to quinolones. Phylogenetic and subtyping assessments showed that highly similar isolates (≥99.4% shared core genome, ≥99.6% shared coding sequence) with priority AMR were found in birds on feedlots separated by distances exceeding 150 km, suggesting that European starlings could be involved in the interstate dissemination of priority AMR bacteria.

Antimicrobial Resistance, Genetic Diversity and Multilocus Sequence Typing of Escherichia coli from Humans, Retail Chicken and Ground Beef in Egypt.

Contamination of retail foods with foodborne pathogens, particularly the antimicrobial resistant ones, poses a persistent threat to human health. There is a dearth of information about the overlapping Escherichia coli (E. coli) lineages circulating among retail foods and humans in Egypt. This study aimed to determine the clonal diversity of 120 E. coli isolates from diarrheic patients (n = 32), retail chicken carcasses (n = 61) and ground beef (n = 27) from Mansoura, Egypt using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Simpson’s index of diversity was calculated to compare the results of both typing methods. Antimicrobial resistance phenotypes, genotypes and phylogrouping of the isolates were also determined. Higher frequencies of antimicrobial resistance were found among chicken isolates compared to beef and human isolates; regardless of isolate source, the predominant antimicrobial resistances were found against ampicillin (87/120, 72.5%), tetracycline and sulfisoxazole (82/120, 68.3%, each), and streptomycin (79/120, 65.8%). None of the isolates displayed resistance to meropenem. The prevalent genes detected were tetA (64.2%), blaTEM (62.5%), sul1 (56.7%), floR (53.3%), sul2 (50%), strB (48.3%) and strA (47.5%) corresponding with resistance phenotypes. Alarmingly, blaCTX was detected in 63.9% (39/61) of chicken isolates. The majority of E. coli isolates from humans (90.6%), beef (81.5%) and chicken (70.5%) belonged to commensal phylogroups (A, B1, C). Using PFGE analysis, 16 out of 24 clusters (66.7%) contained isolates from different sources at a similarity level ≥75%. MLST results assigned E. coli isolates into 25, 19 and 13 sequence types (STs) from chicken, human and beef isolates, respectively. Six shared STs were identified including ST1011, ST156, ST48, ST224 (chicken and beef), ST10 (human and chicken) and ST226 (human and beef). Simpson’s index of diversity was higher for MLST (0.98) than PFGE (0.94). In conclusion, the existence of common genetic determinants among isolates from retail foods and humans in Egypt as well as the circulation of shared STs indicates a possible epidemiological link with potential zoonotic hazards.

Distribution of phenotypic and genotypic antimicrobial resistance and virulence genes in Vibrio parahaemolyticus isolated from cultivated oysters and estuarine water.

A total of 594 Vibrio parahaemolyticus isolates from cultivated oysters (n = 361) and estuarine water (n = 233) were examined for antimicrobial resistance (AMR) phenotype and genotype and virulence genes. Four hundred forty isolates (74.1%) exhibited resistance to at least one antimicrobial agent and 13.5% of the isolates were multidrug-resistant strains. Most of the V. parahaemolyticus isolates were resistant to erythromycin (54.2%), followed by sulfamethoxazole (34.7%) and trimethoprim (27.9%). The most common resistance genes were qnr (77.8%), strB (27.4%) and tet(A) (22.1%), whereas blaTEM (0.8%) was rarely found. Four isolates (0.7%) from oysters (n = 2) and estuarine water (n = 2) were positive to tdh, whereas no trh-positive isolates were observed. Significantly positive associations among AMR genes were observed. The SXT elements and class 1, 2 and 3 integrons were absent in all isolates. The results indicated that V. parahaemolyticus isolates from oysters and estuarine water were potential reservoirs of resistance determinants in the environment. This increasing threat of resistant bacteria in the environment potentially affects human health. A 'One Health' approach involved in multidisciplinary collaborations must be implemented to effectively manage antimicrobial resistance.

Novel diverse sequences of the Streptococcus canis M-like protein (SCM) gene and their prevalence in diseased companion animals: Association of their alleles with sequence types

ObjectiveWe aimed to determine novel alleles and their prevalence in Streptococcus canis M-like protein (SCM) and to elucidate association of their alleles with sequence types (STs)/clonal complexes (CCs) and antimicrobial resistance (AMR) phenotypes/genotypes. MethodsWe amplified and sequenced scm, by using primers reported by Pinho recently, for 40 isolates in 2015 and 2017, in which the sequences could not be determined with conventional primers. Isolates, for which SCM alleles, STs, and AMR phenotypes/genotypes were previously determined, were included as controls. A phylogenetic tree of SCM amino acid sequences was constructed. Alleles, based on the tree positions with their prevalence, as well as STs/CCs and AMR phenotypes/genotypes were characterized. ResultsAlthough one isolate possessed SCM allele type 1, 39 isolates had novel allele types 10–15, based on cluster analysis. The 11 and 12 allele types were firstly found in this study. We designated novel allele types as group II and non-novel allele types as group I. Prevalence of group II alleles was 29.9% and 16.2% in 2015 and 2017. Prevalent group II types were allele 10 (10.3%), allele 11 (2.7%), and allele 15 (3.3%) through both periods. There was a significant difference in distribution of STs/CCs between groups I/II SCM populations. We found significant differences in distribution of macrolide/lincosamide AMR genotype (7.7% vs. 26.8%) and AMR rates of fluoroquinolone (0% vs. 12.5%) between the two populations. ConclusionOur study presents group II scm sequences and their prevalence among diseased companion animals in Japan, with association of their alleles with STs.

Partial Correlation Between Phenotypic and Genotypic Antimicrobial Resistance of Salmonella enterica Serovar Enteritidis Strains from Brazil.

Whole-genome sequencing analyses have provided important data and information on the repertoire of resistance genes in several bacterial species. This study examined to what extent the antimicrobial resistance genes found in a set of whole-genome-sequenced Salmonella Enteritidis strains from Brazil correlated with the phenotypic antimicrobial resistance possibly related to these genes. The genotypic resistance data from the strains studied were compared with publicly available data from strains isolated in other countries. The genotypic resistance profiles were accessed on the NCBI Pathogen Detection website, and the phenotypic resistance profiles were determined by the disk diffusion technique according to the Clinical and Laboratory Standards Institute guidelines. Fourteen of the 256 sequenced strains presented antimicrobial resistance genes, with the highest prevalence of resistance genes to aminoglycosides-with 16 genes detected in seven strains-not only in Brazilian strains but also in the strains from other parts of the world. The strongest correlation between phenotypic and genotypic resistance was found for tetracycline (75%). The genotypic and phenotypic profiles of the S. Enteritidis strains studied only partially matched, and they diverged in some antimicrobial classes more strongly than in other classes. The advances on whole-genome sequencing analyses associated with a better understanding of the correlation between phenotypic and genotypic resistance data may improve this powerful tool for antimicrobial resistance prediction.

Molecular Characterization and Antimicrobial Susceptibility of C. jejuni Isolates from Italian Wild Bird Populations.

Poultry is considered a major reservoir of human campylobacteriosis. It also been reported that not only poultry, but also wild birds, are capable of carrying C. jejuni, thus demonstrating to be a risk of spreading the bacteria in the environment. To gain insight into the population structure and investigate the antimicrobial resistance genotypes and phenotypes, we analyzed a collection of 135 C. jejuni from 15 species of wild birds in Italy. MLST revealed the presence of 41 sequence types (STs) and 13 clonal complexes (CCs). ST-179 complex and the generalist ST-45 complex were the most prevalent. Core genome MLST revealed that C. jejuni from ST-45 complex clustered according to the bird species, unlike the ST-179 complex which featured 3 different species in the same cluster. Overall we found a moderate prevalence of resistance to tetracycline (12.5%), ciprofloxacin and nalidixic acid (10%). The novel ST isolated from one pigeon showed resistance to all the antibiotics tested. The ST-179 complex (33.3%) was identified with significantly higher nalidixic acid resistance relative to other tested STs. Nine AMR genes (tet(O), cmeA, cmeB, cmeC, cmeR, aad, blaOXA-61, blaOXA-184 and erm(B)) and 23S rRNA and gyrA-associated point mutations were also described, indicating a concordance level between genotypic and phenotypic resistance of 23.3%, 23.4% and of 37.5% for streptomycin, tetracycline and quinolones/fluoroquinolones, respectively. We recommend that particular attention should be given to wild birds as key sentinel animals for the ecosystem contamination surveillance.

Draft Genome Sequences of Seven Streptococcus canis Strains Isolated from Diseased Companion Animals in Japan.

The draft genomes of seven Streptococcus canis isolates from diseased dogs and cats in Japan are reported here. The genome sizes ranged from 1.901 Mbp to 2.203 Mbp, with G+C contents of 39.5% to 39.8%. The sequence types, antimicrobial resistance genotypes, and S. canis M-like protein alleles were all characterized.

Antimicrobial Resistance of Commensal Escherichia coli Isolated from Food Animals in Qatar.

Objectives: This study aims at evaluating the phenotypic and genotypic antimicrobial resistance (AMR) patterns of 18 clinically relevant antibiotics in food animals in Qatar. Materials and Methods: Fecal samples from camels, cattle, and pigeons (300) were collected from different slaughterhouses and farms. Escherichia coli isolates were recovered on selective media, confirmed biochemically, and tested for antibiotic susceptibility using a disk diffusion assay. Any isolate that showed resistance to colistin was confirmed using the E-test and polymerase chain reaction for mcr genes. Results: Overall, a total of 88.7% (n = 266/300) recovery rate was achieved from all samples. Resistance to at least one antibiotic was recorded in 70.7% of pigeons, 37.2% of cattle, and only 20.8% of camel samples. Multidrug resistance (MDR) was highest in isolates from pigeons, 50% (n = 44). Moreover, trimethoprim/sulfamethoxazole (an antibiotic used to treat a variety of bacterial infections) resistance was present in 22.2% (n = 59) of all E. coli isolates. Only one E. coli isolate from a pigeon showed resistance to colistin (mcr-1 gene encoded), a drug of last resort in human medicine against gram-negative bacterial infection. Conclusions: We previously reported high multidrug resistance of E. coli in chickens, with significant resistance to colistin. We observed a lower AMR profile in ruminants. The high resistance profile observed in pigeons (70.7%), including high multidrug resistance (50%), is alarming as these animals could rapidly disseminate resistant bacteria to various locations. Continuous monitoring of AMR in livestock in Qatar is necessary toward introducing an antimicrobial stewardship program and control of antibiotic usage in the veterinary sector.

Domestic and game pigeons as reservoirs for Escherichia coli harbouring antimicrobial resistance genes

ObjectivesIn Iran, pigeons are kept and bred on the roofs of houses, which indicates the potential significance of pigeons in the dispersal of antimicrobial resistant Escherichia coli strains. Here we characterized antimicrobial resistance genotypes in relation to phenotypic presentations and phylogenetic backgrounds of the E. coli isolates from household pigeons in Kerman in southeast Iran. MethodsTotally, 152 faecal E. coli isolates from domestic and household pigeons were screened for 13 antimicrobial resistance genes, blaTEM, blaSHV, blaCTX-M, sulI, sulII, dhfrI, dhfrV, aadA, aac(3)-I, tetA, tetB, floR and qnrA, by conventional polymerase chain reaction (PCR) technique. Clermont phylogenetic background of E. coli strains was studied and antibiotic resistance of all strains was assessed for seven antibiotics. ResultsThe antimicrobial resistance genes blaTEM, tetA, tetB and aadA were detected in 52.6%, 6.5%, 6.5% and 5.9% of the isolates, respectively. PCR phylotyping revealed that a significant number of isolates within A0 (54%), A1 (70%), B1 (57.6%), B22 (75%) and D1 (87.5%) phylogroups were positive for the studied resistance genes. One phenotypic resistance pattern (trimethoprim-sulfamethoxazole) was associated with the presence of the corresponding gene sul2. ConclusionsAn alarming rate of phenotypic resistance was observed in this study. Many isolates were positive for the screened resistance genes. According to the phylogenetic background, most resistant isolates belonged to the commensal phylotypes, representing significant role of commensal strains as a source of resistance genes. These findings highlight the role of the pigeon as disseminator of resistant E. coli strains.

Comparison of phenotypic and genotypic antimicrobial resistance patterns associated with Staphylococcus aureus mastitis in German and Danish dairy cows

Staphylococcus aureus is one of the most common pathogens associated with bovine mastitis in Germany and Denmark. Successful therapy is strongly linked to the susceptibility of the pathogen to the administered antimicrobial. An increase in resistant pathogens in human and veterinary medicine has become a concern worldwide and hampers therapy due to reduced susceptibility. In the present study, susceptibility testing was performed for 85 and 93 S. aureus isolates originating from mastitis cases on 12 German and 8 Danish dairy farms, respectively. Phenotypic examination was performed by detection of minimal inhibitory concentration (MIC) values using the broth microdilution method, followed by genotypic investigations of the blaZ and mecA resistance genes via PCR. The tested antimicrobials were the most frequently used β-lactams in German and Danish dairy farms, including cefquinome, cefoperazone, cephapirin, penicillin, oxacillin, cloxacillin, amoxicillin-clavulanic acid, and cephalexin-kanamycin. Special attention was paid to varying therapy concepts because, in Germany, third- and fourth-generation cephalosporins have been predominantly used in mastitis therapy, whereas in Denmark, restrictive use of penicillin is followed by a general avoidance of cephalosporins. Differences in MIC values were mainly based on determined MIC90 values (MIC at which 90% of isolates are inhibited). In general, Danish S. aureus isolates were inhibited at comparatively lower MIC90 values than S. aureus isolated from German dairy farms for most β-lactams. No differences were observed regarding cefquinome, because both German and Danish isolates exhibited MIC50 and MIC90 values of 0.5 and 1 µg/mL, respectively. In contrast, the MIC90 for penicillin against German and Danish S. aureus were 0.5 and ≤0.06 µg/mL, respectively. Resistance genes (blaZ, mecA) were only detected in German S. aureus isolates on 3 dairy farms in Germany. A total of 5 isolates tested positive for both blaZ and mecA, whereas 1 isolate carried the blaZ resistance gene only. A direct correlation between frequently used antimicrobials and reduced susceptibility could not be determined based on results of the present study. In addition to further research to determine factors associated with resistance development, we emphasize the urgent need for internationally standardized clinical breakpoints to assess resistance situations more accurately.

Mycoplasma genitalium Antimicrobial Resistance in Community and Sexual Health Clinic Patients, Auckland, New Zealand

Our retrospective study compared genotypic antimicrobial resistance in Mycoplasma genitalium–positive specimens collected from 48 community and 33 sexual health clinic (SHC) patients. Macrolide resistance was similar in community (75%) and SHC (76%) patients. We observed no significant difference in fluoroquinolone resistance between community (19%) and SHC (27%) patients (p = 0.66).

Multidrug-Resistant Vibrio species recovered from some Freshwater Resources in Southwest Nigeria: a public health concern

Globally, pathogens that cause infectious water-related diseases such as diarrhea and cholera are beginning to manifest with unusual antibiotic resistance characteristics and virulence, posing a huge threat to public health. Vibrio species are a leading cause of water-and food-borne outbreaks and is widely distributed in the water environments making it a significant threat to human health worldwide. A total of 315 Vibrioisolates recovered from different freshwater resources in southwest Nigeria were confirmed by simplex PCR using toxR gene. All confirmed isolates were tested for In vitro susceptibility to 18 antibiotics using agar disc-diffusion assay and the phenotypic tetracycline-resistant isolates were further profiled for their genotypic antimicrobial resistance determinants by PCR assay. The isolates were variously susceptible to the antibiotics tested as follows: norfloxacin 308 (98%), ciprofloxacin 293 (93%), meropenem 287 (91%), cefotaxime 279 (88%), amikacin 238 (75%). Conversely, resistance to erythromycin 300 (95%), sulphamethoxazole 297 (94%), rifampicin 289 (92%), doxycycline 260 (82%), tetracycline 237 (75%) were equally observed. Tetracycline resistance isolates were assessed for resistance determinant and the following prevalence were obtained; tetA (28), tetE (20), and tet39 (3). The MAR index across the sampling locations of 0.8 to 0.94 exceeds the threshold value of 0.2, suggesting excessive antimicrobial usage at the sample source. Our findings reveal high incidence ofVibrio species in the selected freshwater resources and also signify high resistance towards some conventionally used antibiotics. Consequently, this portends that the waters are unfit for domestic, industrial and recreational purposes and a reservoir of antibiotic resistance determinants in the environment.

Antimicrobial resistance phenotypes and genotypes of methicillin-resistant Staphylococcus aureus CC398 isolates from Spanish hospitals

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of lineage CC398 is an emerging clone causing human infections but is mostly found in pigs. The aim of this study was to characterize the antimicrobial resistance phenotypes/genotypes of a collection of 137 MRSA CC398 isolates obtained in a previous study from 17 Spanish hospitals, using tetracycline resistance as marker for selection. A multidrug-resistant (MDR) phenotype was present in 79% of analysed isolates, with 17% of them resistant to at least six different antimicrobial families. All tetracycline-resistant isolates (n=137) carried the tetM gene and 75% also carried the tetK gene. Almost 50% of MRSA CC398 isolates showed macrolide and/or lincosamide resistance: a) 39% of isolates were ERYR-CLIR (all with constitutive phenotype), with 87% of them carrying the ermC gene, followed by msrA (25%), ermB (21%), vgaA (17%), ermA (6%), lsaB (4%), linA (2%), linB (2%), and ermT (2%, this isolate with the new spa-type t18071); and b) 9% of MRSA CC398 isolates showed the dissociated ERYS-CLIR phenotype carrying the linA, linB, lsaB and vgaA genes. Other antimicrobial resistance phenotypes in these MRSA CC398 isolates included resistance to ciprofloxacin (67%), aminoglycosides (21%), mupirocin (6%), chloramphenicol (4%) or fusidic acid (2%). The more common resistance genes detected for some of these antimicrobials were: aac(6’)-Ie-aph(2’’)-Ia (16%) and ant(4’)-Ia (12%) for aminoglycosides, and fexA (3%) for chloramphenicol. The high rate of MDR phenotypes with a wide range of antimicrobial resistance genes shown in this study reduce the potential therapeutic options in case of infections.

MOLECULAR CHARACTERIZATION OF FECAL ESCHERICHIA COLI ISOLATED FROM ZOO ANIMALS.

Accredited zoos and animal parks play an important role in animal health research and conservation, providing important insights on matters of public health including zoonotic infectious diseases and antimicrobial resistance (AMR). The emergence and spread of AMR is a complex phenomenon that jeopardizes human and animal health and also threatens the long-term survival of endangered species. The presence of β-lactamases in clinical isolates is particularly significant as they can jeopardize the efficacy of critically important antimicrobials. Although the presence of β-lactamases and extended-spectrum β-lactamases (ESBLs) producing Enterobacteriaceae in zoo animals has been reported, data are not available for northern European countries. In addition, few data are available on phylogenetic grouping of Escherichia coli isolated from zoo animals that can provide additional information on the host-bacterium relationship and on the pathogenicity of isolates. This study aimed to characterize fecal E. coli isolated from 33 healthy zoo animals from 22 species in Ireland, using conventional and molecular microbiological methods. All E. coli isolates were ampicillin resistant, but combined resistance to amoxicillin and clavulanic acid was not detected. Three E. coli isolates sampled from one Amur tiger, one Bornean orangutan, and one Southern white rhino were multidrug resistant, and blaTEM was detected in E. coli recovered from the Amur tiger and the Bornean orangutan. Other β-lactamases, including ESBLs and AmpCs and plasmid-mediated mcr-1 and mcr-2, were not detected. Overall, E. coli isolates investigated were susceptible to the majority of the antimicrobials tested, and only two animals shed E. coli carrying β-lactamase-encoding genes. The majority of isolates belonged to phylogenetic group B1. The screening of the AMR phenotype and genotype of zoo animal E. coli provides useful data that is relevant to antimicrobial stewardship in the zoo veterinary services and relevant to the bank of knowledge needed for tackling AMR.

Repetitive cellulitis caused by Streptococcus agalactiae isolates with different genotypic and phenotypic features in a patient having upper extremity with lymphedema after mastectomy and axillary lymph node dissection

Previously reported cases of recurrent cellulitis/erysipelas affecting chronically lymphedematous skin regions have been demonstrated to be due to Streptococcus agalactiae isolates with closely related genetic background which may be suggestive of relapse rather than reinfection. Herein, we report the occurrence of three episodes of repetitive cellulitis caused by S. agalactiae strains with different genotypic and phenotypic characteristics, including different antimicrobial susceptibility patterns (tetracycline, macrolide/lincosamide, and fluoroquinolone classes), in the left upper extremity of a patient with lymphedema, following left mastectomy and axillary lymph node dissection. The genotypic and phenotypic characteristics of the three isolates were confirmed based on the random amplified polymorphic DNA patterns, DNA profiles of virulence factors (bca-rib-bac-lmb-cylE), data on biofilm formation and cell invasion, antimicrobial susceptibility testing results, antimicrobial resistance (AMR) genotypes, and amino acid mutations associated with AMR. These results revealed that reinfection with S. agalactiae, rather than recurrence, occurred during the three episodes. In conclusion, microbiologic studies such as blood cultures or tissue cultures are certainly helpful in the management of recurrent infections or invasive infections such as bacteremia in order to better target antimicrobial therapy, regardless of the data previously presented.