blaZ Gene Research Articles

First comparative genomic characterization of the MSSA ST398 lineage detected in aquaculture and other reservoirs.

Staphylococcus aureus ST398 can cause diseases in several different animals. In this study we analyzed ten S. aureus ST398 previously collected in three different reservoirs in Portugal (humans, gilthead seabream from aquaculture and dolphin from a zoo). Strains tested against sixteen antibiotics, by disk diffusion or minimum inhibitory concentration, showed decreased susceptibility to benzylpenicillin (all strains from gilthead seabream and dolphin) and to erythromycin with an iMLSB phenotype (nine strains), and susceptibility to cefoxitin (methicillin-susceptible S. aureus, MSSA). All strains from aquaculture belonged to the same spa type, t2383, whereas strains from the dolphin and humans belonged to spa type t571. A more detailed analysis using single nucleotide polymorphisms (SNPs)-based tree and a heat map, showed that all strains from aquaculture origin were highly related with each other and the strains from dolphin and humans were more distinct, although they were very similar in ARG, VF and MGE content. Mutations F3I and A100V in glpT gene and D278E and E291D in murA gene were identified in nine fosfomycin susceptible strains. The blaZ gene was also detected in six of the seven animal strains. The study of the genetic environment of erm(T)-type (found in nine S. aureus strains) allowed the identification of MGE (rep13-type plasmids and IS431R-type), presumably involved in the mobilization of this gene. All strains showed genes encoding efflux pumps from major facilitator superfamily (e.g., arlR, lmrS-type and norA/B-type), ATP-binding cassettes (ABC; mgrA) and multidrug and toxic compound extrusion (MATE; mepA/R-type) families, all associated to decreased susceptibility to antibiotics/disinfectants. Moreover, genes related with tolerance to heavy metals (cadD), and several VF (e.g., scn, aur, hlgA/B/C and hlb) were also identified. Insertion sequences, prophages, and plasmids made up the mobilome, some of them associated with ARG, VF and genes related with tolerance to heavy metals. This study highlights that S. aureus ST398 can be a reservoir of several ARG, heavy metals resistance genes and VF, which are essential in the adaption and survival of the bacterium in the different environments and an active agent in its dissemination. It makes an important contribution to understanding the extent of the spread of antimicrobial resistance, as well as the virulome, mobilome and resistome of this dangerous lineage.

Inhibition Mechanism of Methicillin-Resistant Staphylococcus aureus by Zinc Oxide Nanorods via Suppresses Penicillin-Binding Protein 2a.

Methicillin-resistant Staphylococcus aureus (MRSA) causes life-threatening infections. Zinc oxide is well known as an effective antibacterial drug against many bacterial strains. We investigated the performance of zinc oxide nanorods synthesized by Albmiun as a biotemplate as an antibacterial drug in this study; the fabrication of zinc oxide nanorods was synthesized by sol-gel methods. We performed physicochemical characterization of zinc oxide nanorods by physiochemical techniques such as FTIR spectroscopy, X-ray diffraction, and TEM and investigation of their antimicrobial toxicity efficiency by MIC, ATPase activity assay, anti-biofilm activity, and kill time assays, as well as the mecA, mecR1, blaR1, blaZ, and biofilm genes (ica A, ica D, and fnb A) by using a quantitative RT-PCR assay and the penicillin-binding protein 2a (PBP2a) level of MRSA by using a Western blot. The data confirmed the fabrication of rod-shaped zinc oxide nanorods with a diameter in the range of 50 nm, which emphasized the formation of zinc oxide nanoparticles with regular shapes. The results show that zinc oxide nanorods inhibited methicillin-resistant S. aureus effectively. The MIC value was 23 μg/mL. The time kill of ZnO-NRs against MRSA was achieved after 2 h of incubation at 4MIC (92 μg/mL) and after 3 h of incubation at 2MIC (46 μg/mL), respectively. The lowest concentration of zinc oxide nanorods with over 75% biofilm killing in all strains tested was 32 μg/mL. Also, we examined the influence of the zinc oxide nanorods on MRSA by analyzing mecA, mecR1, blaR1, and blaZ by using a quantitative RT-PCR assay. The data obtained revealed that the presence of 2× MIC (46 μg/mL) of ZnO-NRs reduced the transcriptional levels of blaZ, blaR1, mecA, and mecR1 by 3.4-fold, 3.6-fold, 4-fold, and 3.8-fold, respectively. Furthermore, the gene expression of biofilm encoding genes (ica A, ica B, ica D, and fnb A) was tested using quantitative real-time reverse transcriptase-polymerase chain reaction (rt-PCR). The results showed that the presence of 2× MIC (46 μg/mL) of ZnO-NRs reduced the transcriptional levels of ica A, ica B, ica D, and fnb A. Also, the PBP2a level was markedly reduced after treatment with ZnO-NRs.

Phenotypic and Genotypic Characterization of Macrolide-Lincosamide-Streptogramin Resistance in Staphylococcus aureus Isolates from Bovine and Human

Abstract In this study, penicillin, oxacillin, and macrolide-lincosamide-streptogramin (MLS) resistance in S. aureus strains that were isolated from bovine mastitis cases, and human patients were investigated. Inducible clindamycin resistance (iML) was not found in 30 bovine isolates, while it was detected in 3 (10%) of 30 human isolates. MIC90 values of penicillin, oxacillin and macrolide-lincosamides (ML) were 2, 0.19, >256 µg/ml in bovine isolates and were 3, 3 and 0.19-1.5 µg/ml in human isolates, respectively. Streptogramin resistance was not found in both bovine and human isolates. Although the mecA gene was detected in all of the oxacillin resistant isolates, blaZ gene could not be detected in penicillin resistant isolates. The erm(B) gene was detected in 5 (38.6%) of 13 ML-resistant bovine isolates, and the mph(C) gene was detected in 2 (66.66%) of 3 human isolates. As a result, resistance to penicillin and oxacillin was found to be higher in human S. aureus isolates, while ML resistance was found to be higher in bovine isolates in this investigation. It was concluded that the presence of genes in extra-chromosomal elements associated to penicillin and macrolide resistance should be investigated. The data obtained from this study will contribute to the studies on antimicrobial susceptibility in the field of human and veterinary medicine.

PREVALENCE, PHENOTYPIC-GENOTYPIC RESISTANCE AND BIOFILM FORMATION OF Staphylococcus aureus IN CHICKEN MEAT WITH REFERENCE TO ITS PUBLIC HEALTH HAZARD

The purpose of this study was to determine the prevalence, antimicrobial resistance in S. aureus and presence of enterotoxigenic as well as biofilm-forming genes in S. aureus bacteria isolated from broiler meat from traditional shops and supermarkets in Sharkia, Egypt. For this determination, two hundred fresh raw chicken meat cotton swabs were collected from the breast and thighs (100 each). S. aureus was highlighted through a coagulase test, and then phenotypic and genotypic characterizations were studied. Uniplex PCR was used to identify the occurrence of enterotoxin genes in the selected isolates. Finally, they were subjected to a biofilm formation test using 96-well flat bottom polystyrene microtiter plates; besides, biofilm-forming genes were investigated. Nineteen isolates out of the 200 samples tested positive for S. aureus (9.5 percent), No Vancomycin resistance strains were obtained, nor did any ciprofloxacin isolates for S. aureus while, all isolates were 100 % resistant for streptomycin, amoxicillin- clavulanic acid and sulfamethoxazole-trimethoprim S. aureus isolates were found resistant to at least one antibiotic. The multiple antibiotic resistance (MAR) index of S. aureus isolates was ranged from 0.3 to 0.7 with an average of 0.4. In The mecicillin resistant S.aureus (MRSA) strains carried mecA gene with a percentage of 5%, while the blaZ gene was distributed with a percentage of 5.5%, 11/200). The obtained isolates gave 60% strong biofilm formation and 40% were non-formers, with nil results for moderate or weak production isolates. The icaA gene was 100%, in comparison to icaD which was zero. It should be noted that strong biofilm former strains were only 100% positive for sea and seb. This study pointed out the higher prevalence of MDR isolates of S. aureus isolates in chicken meat due to inadequate handling and insufficient sanitary equipment and post microbial contamination. This finding highlighted the importance of broiler meat as a reservoir for antimicrobial-resistant strains of S. aureus and biofilm-forming of Staphylococcus aureus.

Recent Trends of Antibiotic Resistance in Staphylococcus aureus Causing Clinical Mastitis in Dairy Herds in Abruzzo and Molise Regions, Italy.

This study aimed to investigate the recent trends of antibiotic resistance (AR) prevalence in Staphylococcus aureus isolated from the milk of animals with clinical mastitis in areas of the Abruzzo and Molise regions in Central Italy. Fifty-four S. aureus isolates were obtained from routine testing for clinical mastitis agents carried out in the author institution in the years 2021 and 2022 and were analyzed for phenotypic resistance to eight antibiotics recommended for testing by European norms and belonging to the antibiotic classes used for mastitis treatment in milk-producing animals. Moreover, the presence of 14 transferable genetic determinants encoding resistance to the same antibiotics was analyzed using qPCR tests developed in this study. Phenotypic resistance to non-β-lactams was infrequent, with only one 2022 isolate resistant to clindamycin. However, resistance to the β-lactam cefoxitin at concentrations just above the threshold of 4 µg/mL was observed in 59.2% of isolates in both years, making these isolates classifiable as methicillin-resistant. The AR genotypes detected were the blaZ gene (50% of 2021 isolates and 44.4% of 2022 isolates), aphA3-blaZ- ermC/T (one 2021 isolate), aphA3-ant6-blaZ-ermC/T (one 2021 isolate), blaZ-ermB (one 2022 isolate) and mecA-mph (one 2022 isolate). An inquiry into the veterinarians who provided the samples, regarding the antimicrobials prescribed for mastitis treatment and criteria of usage, indicated a possible causal relation with the AR test results. The occurrence of AR genotypes did not increase in time, most probably reflecting how mastitis was treated and prevented in farms. However, the frequently observed cefoxitin resistance needs to be explained genotypically, further monitored and limited by modifying antibiotic usage practices. The identification of a mecA-positive isolate in 2022 suggests further investigation if this genotype is emerging locally.

Genomic Evidence for Direct Transmission of mecC-MRSA between a Horse and Its Veterinarian

Methicillin-resistant Staphylococcus aureus bearing the mecC gene (mecC-MRSA) has been reported from animals and humans in recent years. This study describes the first mecC-MRSA isolates of human and equine origin in Hungary (two isolates from horses and one from a veterinarian, who treated one of the infected horses, but was asymptomatic). MRSA isolates were identified by cultivation and PCR detection of the species-specific spa gene and mecA/mecC methicillin resistance genes. The isolates were characterized by antibiotic susceptibility testing, MLST, spa, SCCmec typing, PFGE and whole genome sequencing (WGS). All three isolates belonged to the ST130-t843-SCCmec XI genotype, and carried the mecC and blaZ genes. Apart from beta-lactam drugs, they were sensitive to all tested antibiotics. The isolates of the infected horse and its veterinarian had the same PFGE pulsotype and showed only slight differences with WGS. Hence, this is the first description of direct transmission of a mecC-carrying MRSA between a horse and its veterinarian. The emergence of mecC in the country highlights the importance of the appropriate diagnostics in MRSA identification.

Association of multilocus sequencing types and antimicrobial resistance profiles of methicillin-resistant Mammaliicoccus sciuri in animals in Southern Thailand.

Mammaliicoccus sciuri, formerly known as Staphylococcus sciuri, is an opportunistic pathogen in the environment, human and animal mucosa, and skin. Although this pathogen is becoming more resistant to drugs and harmful to animals and humans, basic knowledge of this pathogen remains limited. This study aimed to investigate a new multilocus sequencing type (MLST) related to the antibiotic resistance pattern of M. sciuri from animals in southern Thailand. We used 11 methicillin-resistant M. sciuri (MRMS) isolates in this study which were obtained from six horses, four cows, and one chicken of the previous study. Antimicrobial resistance (AMR) was re-evaluated based on the minimum inhibitory concentration using the VITEK® 2 automated system. Three AMR genes were examined, namely mecA, mecC, and blaZ. Staphylococcal chromosomal cassette mec (SCCmec) gene detection was performed through the multiplex polymerase chain reaction (PCR). Internal segments of the seven housekeeping genes, ack, aroE, ftsZ, glpK, gmk, pta1, and tpiA, were used for multilocus sequence typing. The population of resistant bacteria and the types of multidrug-resistant, extensively drug-resistant, and pandemic drug-resistant bacteria were classified through descriptive analysis. mecA and blaZ genes were detected in all isolates; however, the mecC gene was not observed in any isolate based on the PCR results. All MRMS isolates revealed a non-typable SCCmec. Seven MLSTs (71, 81, 120, 121, 122, 199, and 200) were identified in this study. The characteristics of MRMS in Southern Thailand were variable, particularly in cattle and horses. The antibiogram and SCCmec types of this pathogen remain concerns with regard to antibiotic-resistant gene transmission among Staphylococcus and Mammaliicoccus species. All MLSTs in Thailand revealed the distribution among clones in Asia, including the virulence of a zoonotic clone in Southern Thailand.

Antibiotic resistance of probiotics isolated from Chinese corn stover silage

ABSTRACT One of the most important drivers of the emergence of antimicrobial resistance is the irrational usage of antibiotics. But alongside some indirect sources such as feeding Chinese corn stover silage could also transmit resistant genes to animals. This study was aimed at assessing the antibiotic resistance phenotypes, drug resistance genes and mediated drug resistance mechanisms. A total of 37 isolates were obtained by selective medium and identified by 16S rDNA sequencing including Lactobacillus acidophilus (n = 3), Lactobacillus amylovorus (n = 4), Weissella confusa (n = 3), Acetobacter pasteurianus (n = 9), Lactobacillus buchneri (n = 6), Enterococcus faecium (n = 8) and Lactobacillus reuteri (n = 4). Antimicrobial resistance of all isolates to 12 antibiotics was determined using the agar dilution method. Widespread resistance to ampicillin, erythromycin, clindamycin, kanamycin, streptomycin, levofloxacin and ciprofloxacin was observed. The presence of relevant resistance genes was examined by PCR, and the genes pbp5, blaZ, bla TEM, mecA, mexI, tetW, ermB, msrA, msrC, ermC, vatE, aacA-aphD, aphA1, aadA1, aadA2 and vanX were detected. All the isolated strains had multiple drug resistance, and harboured related drug-resistant genes; therefore, the use of probiotics in animal feed should be standardized to reduce the risks of horizontal transmission of drug resistance genes to animal products and human population. Highlights Fermentation bacteria isolated from the silage were identified. Lactobacillus strains isolated from the silage exhibited antimicrobial resistance. Feeding Chinese corn stover silage transmits resistant genes to animals. Standardization of probiotics in animal feed is crucial to avoid horizontal drug resistance.

Molecular Characterizations of the Coagulase-Negative Staphylococci Species Causing Urinary Tract Infection in Tanzania: A Laboratory-Based Cross-Sectional Study

There is a growing body of evidence on the potential involvement of coagulase-negative Staphylococci (CoNS) in causing urinary tract infections (UTIs). The aim of this study was to delineate virulence potential, antimicrobial resistance genes, and sequence types of CoNS isolated from patients with UTI symptoms and pyuria in Tanzania. CoNS from patients with UTI symptoms and more than 125 leucocytes/μL were retrieved, subcultured, and whole-genome sequenced. Out of 65 CoNS isolates, 8 species of CoNS were identified; Staphylococcus haemolyticus, n = 27 (41.5%), and Staphylococcus epidermidis, n = 24 (36.9%), were predominant. The majority of S. haemolyticus were sequence type (ST) 30, with 8 new ST138-145 reported, while the majority of S. epidermidis were typed as ST490 with 7 new ST1184-1190 reported. Sixty isolates (92.3%) had either one or multiple antimicrobial resistance genes. The most frequently detected resistance genes were 53 (21%) dfrG, 32 (12.9%) blaZ, and 26 (10.5%) mecA genes conferring resistance to trimethoprim, penicillin, and methicillin, respectively. Out of 65 isolates, 59 (90.8%) had virulence genes associated with UTI, with a predominance of the icaC 47 (46.5%) and icaA 14 (13.9%) genes. Conclusion:S. haemolyticus and S. epidermidis harboring icaC, dfrG, blaZ, and mecA genes were the predominant CoNS causing UTI in Tanzania. Laboratories should carefully interpret the significant bacteriuria due to CoNS in relation to UTI symptoms and pyuria before labeling them as contaminants. Follow-up studies to document the outcome of the treated patients is needed to add more evidence that CoNS are UTI pathogens.

Occurrence of virulence genes and methicillin-resistance in Staphylococcus aureus isolates causing subclinical bovine mastitis in Tiaret area, Algeria.

Mastitis remains the most frequent and the most expensive disease of dairy breeding. The objective of the study was to study S. aureus isolates collected from subclinical bovine mastitis in the Tiaret region, Algeria, by determining their antimicrobial susceptibility and their virulence traits. Sixty-two S. aureus isolates collected from subclinical bovine mastitis were studied by determining their antimicrobial susceptibility according to CLSI guidelines, and nine genes encoding virulence factors and resistance to methicillin and penicillin were determined by PCR. Multi-drug resistance was observed in 19 (30.64%) isolates and five (8%) were methicillin-resistant S. aureus (MRSA), four of them harbored the mecA gene; however, the mecC gene was not detected. Out of 59 penicillin-resistant isolates, 14 harbored the blaZ gene; one of them co-harbored the mecA gene. The following virulence genes were detected: eta (n=23; 37%), icaA (20; 32.2%), icaD (18; 29%), etb (16; 25.8%), luk E-D (14; 22.5%), and sea (6; 9.6%). The tsst-1, lukF/lukS, and luk-M genes were not detected. The occurrence of MRSA and multi-drug resistant (MDR) S. aureus isolates as well as genes encoding virulence factors playing an important role in the pathogenesis of subclinical bovine mastitis and of harmful potential to human is a cause for concern.

Isolation and Determination of Antibacterial Sensitivity Characteristics of Staphylococcus aureus from Lactating Cows in West Shewa Zone, Ethiopia.

Staphylococcus (S.) aureus is one of the etiologies of bovine mastitis, hindering milk production and productivity in dairy farms. This study was aimed at assessing the distribution of bovine mastitis and the isolation rate of S. aureus in milked cows of West Shewa Zone. The clinical mastitis was diagnosed by physical methods including observation and palpation, whereas the subclinical mastitis was tested by the California mastitis test (CMT). All of the cows tested for mastitis were aseptically sampled (teat-milk) for bacteriology. The bacterium was primarily identified based on colony characterization, catalase, coagulase tests, and Gram stain reaction. Finally, MALDI-TOF Biotyper confirmed the species. The antibacterial sensitivity characteristics of the isolates to different antibacterial drugs were tested by the disk diffusion method. The drugs were selected based on the frequent usage in veterinary medicine in the study area. By using particular primers, the presence of the resistance (mecA and blaZ), and thermonuclease (nuc) genes were determined by polymerase chain reaction (PCR). The data were analyzed by R statistical software. The associations between the dependent variables (prevalence of mastitis and S. aureus) and the explanatory variables were analysed by chi-square (χ 2) and logistic regression tests at a 95% confidence interval (CI). Accordingly, 258 lactating cows were examined, of which 97 (37.6%) were mastitis positive. Of these mastitis positive cows, 59 (60.8%) were subclinical and 38 (39.2%) were clinical. Among the 258 milk samples, 43 (16.7%) were positive for S. aureus. According to the results of the current investigation, subclinical mastitis was significantly more prevalent than clinical mastitis (p < 0.05). The disease was found varied with the lactation stage of the animal, milking with washed hand, udder washing before milking, and tick infestation of the teat. In comparison to animals from farms with lower number of lactating cows, the prevalence of the bacteria was significantly higher in animals managed in farms with large (OR = 12.58, 95% CI = 2.33-68.54, and p < 0.05) and medium (OR = 12.58, 95% CI = 2.33-68.54, and p < 0.05) population of lactating cows per herd. The isoation rate of the bacterium was also found significantly higher in tick-infested cows (OR = 27.69, 95% CI = 9.71-93.01, and p < 0.05) than tick free cows. The antibiogram tests revealed that the isolates resisted penicillin G and tetracycline group drugs (oxytetracycline and tetracycline). Moreover, the nuc gene was confirmed to be present in all of the examined isolates. However, they were not found harboring blaZ and mecA genes. We concluded that S. aureus is sustaining as a main causative agent of bovine mastitis, and they were resistant to the frequently used antibiotics in public and veterinary medicines in the study areas. Therefore, research-based interventions need to be taken in action to combat the pathogen.

Characterisation of Methicillin-Resistant Staphylococcus aureus from Alexandria, Egypt.

The present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt's second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+fus] (n = 6), CC1-MRSA-[V+fus+tir+ccrA/B-1] (PVL+) (n = 5) as well as CC1-MRSA-[V+fus+tir+ccrA/B-1] and CC1153-MRSA-[V+fus] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCCmec V or VI elements that included the fusidic acid resistance gene fusC. The SCCmec [V+fus+tir+ccrA/B-1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ, aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCCmec elements comprising mecA and fusC. This is an unsettling trend, but more MRSA typing data from Egypt are required.

Diversity, heavy metals, and antibiotic resistance in culturable heterotrophic bacteria isolated from former lead-silver-zinc mine heap in Tarnowskie Gory (Silesia, Poland).

Mine tailings represent a great environmental concern due to their high contents of heavy metals. Cultivation analysis of microbiota of Tarnowskie Góry (Poland) mine tailing showed the occurrence of bacteria with colony-forming units as low as 5.7 × 104 per one gram of dried substrate. Among 110 bacterial isolates identified by a combination of MALDI-TOF mass spectrometry and 16S rRNA gene sequencing, phylum Actinobacteria was dominant, followed by Firmicutes and Proteobacteria. Extremely high levels of heavy-metal resistance were observed in Arthrobacter spp., particularly for zinc (500mg/L), lead (1500mg/L), and cadmium (1000mg/L). On the other hand, Staphylococcus spp. showed high tolerance to several antibiotics tested, especially ampicillin, partly due to blaZ gene presence. Due to the occurrence of antibiotic-resistant bacteria, mine tailings are not the cause of heavy-metal contamination only, but also a source of antibiotic-resistant bacteria and thus may represent a serious risk for public health.

Occurrence of antimicrobial resistance and antimicrobial resistance genes in methicillin-resistant Staphylococcus aureus isolated from healthy rabbits.

Methicillin-resistant globally, Staphylococcus aureus (MRSA) is a major cause of disease in both humans and animals. Several studies have documented the presence of MRSA in healthy and infected animals. However, there is less information on MRSA occurrence in exotic pets, especially healthy rabbits. This study aimed to look into the antimicrobial resistance profile, hidden antimicrobial-resistant genes in isolated bacteria, and to estimate prevalence of MRSA in healthy rabbits. Two-hundreds and eighteen samples, including 42 eyes, 44 ears, 44 oral, 44 ventral thoracic, and 44 perineal swabs, were taken from 44 healthy rabbits that visited the Prasu-Arthorn Animal Hospital, in Nakornpathom, Thailand, from January 2015 to March 2016. The traditional methods of Gram stain, mannitol fermentation, hemolysis on blood agar, catalase test, and coagulase production were used to confirm the presence of Staphylococcus aureus in all specimens. All bacterial isolates were determined by antimicrobial susceptibility test by the disk diffusion method. The polymerase chain reaction was used to identify the antimicrobial-resistant genes (blaZ, mecA, aacA-aphD, msrA, tetK, gyrA, grlA, and dfrG) in isolates of MRSA with a cefoxitin-resistant phenotype. From 218 specimens, 185 S. aureus were isolated, with the majority of these being found in the oral cavity (29.73%) and ventral thoracic area (22.7%), respectively. Forty-seven (25.41%) MRSAs were found in S. aureus isolates, with the majority of these being found in the perineum (16, 34.04%) and ventral thoracic area (13, 27.66%) specimens. Among MRSAs, 29 (61.7%) isolates were multidrug-resistant (MDR) strains. Most of MRSA isolates were resistant to penicillin (100%), followed by ceftriaxone (44.68%) and azithromycin (44.68%). In addition, these bacteria contained the most drug-resistance genes, blaZ (47.83%), followed by gyrA (36.17%) and tetK (23.4%). This study revealed that MRSA could be found even in healthy rabbits. Some MRSAs strains were MDR-MRSA, which means that when an infection occurs, the available antibiotics were not effective in treating it. To prevent the spread of MDR-MRSA from pets to owners, it may be helpful to educate owners about effective prevention and hygiene measures.

Whole Genome Sequencing of a Clinical Methicillin-Resistant Staphylococcus aureus from Terengganu Led to the Discovery of a Novel 58.4 kb Conjugative Plasmid

Methicillin-resistant Staphylococcus aureus (MRSA) has been listed by the World Health Organization as a priority antibiotic-resistant nosocomial pathogen in urgent need of new antimicrobials [1]. Plasmids are a key factor in the pathology and epidemiology of MRSA isolates and play important roles in their evolution. In our on-going efforts to better understand the genetic background and molecular epidemiology of MRSA isolates from Terengganu, Malaysia, whole genome sequencing using Illumina HiSeq platform (HiSeq-PE150) was performed on a 31 selection of clinical isolates obtained from the main tertiary hospital, Hospital Sultanah Nur Zahirah (HSNZ). One of these isolates, designated S. aureus SauR23, was isolated from pus in 2016 and was resistant to seven classes of antimicrobials and thus categorized as multidrug resistant (MDR). Analysis of the genome sequence of SauR23 showed it belonged to the ST22 clone with a SCCmec class IV(2B) and harbored the blaZ, mecA, norA, lmrS, mepR and ermC resistance genes, which corresponded to its MDR phenotype. Besides the norA-encoded efflux pump, fluoroquinolone resistance in SauR23 could also be due to the S84L point mutation in its gyrA-encoded DNA gyrase. SauR23 was found to carry three plasmids, designated pSauR23-1 (58,422 bp), pSauR23-2 (3,011 bp), and pSauR23-3 (2,473 bp). The smallest plasmid, pSauR23-3 was a RepL-family plasmid that encoded the ermC gene and was likely responsible for the inducible macrolide-lincosamide-streptogrammin B (iMLSB) phenotype exhibited by the host strain. Plasmid pSauR23-2 was a Rep_1 family small cryptic plasmid. On the other hand, pSauR23-1 is a novel, potentially conjugative plasmid that contains a replicase of the RepA_N domain (repUS20) that shared low sequence identity (34%) to the RepA_N replicase of pWBG749, a known staphylococcal conjugative plasmid. The putative conjugative region of pSauR23-1 differed from the conjugative transfer system of pWBG749 as well as other known conjugative systems in the database. Nevertheless, genes which contained conserved motifs for the MobL relaxase, the TraG/TraD-like conjugative transfer protein, and a type VI secretion system ATPase were detected within this region of the plasmid.

Genomic analysis of a multidrug methicillin-resistant staphylococcus epidermidis recovered from the urine of a guinea pig (Cavia porcellus) with suspected pyelonephritis.

Staphylococcus epidermidis is an emerging pathogen causing infant pyelonephritis. There is a lack of genomic data on Staphylococcus epidermidis as the etiology of pyelonephritis and its resistant determinants. In this study, we have conducted a genomic and microbiologic investigation of an S. epidermidis recovered from the urine of a guinea pig with suspected pyelonephritis in Brazil. The genome was sequenced using the Illumina MiSeq platform and de novo assembled using SPades. Resistome, virulome, and plasmidome were in silico predicted using bioinformatics tools. Data analysis revealed that S. epidermidis USP-LZB-G06 belonged to sequence type ST332. Strikingly, a broad resistome (antibiotics, hazardous heavy metals, and biocides) was predicted, including the presence of the clinically relevant mecA, blaZ, and qacA efflux pump genes. SNP-based analysis revealed that strain USP-LZB-G06 was clustered along mecA positive S. epidermidis strains of ST332 isolated between 2008 and 2016 from humans in Australia and the United States of America. Our results indicate that the detection of this microorganism should be considered as a urinary tract infection agent in exotic pets, particularly guinea pigs. In addition, there is an urgent need to update veterinarians regarding the detection and therapeutic management of these microorganisms.

Relationship between Biofilm Formation and Antibiotic Resistance and Adherence Genes in Staphylococcus aureus Strains Isolated from Raw Cow Milk in Shahrekord, Iran

The production of biofilms by S. aureus contributes significantly to treatment failures. The present study aims to establish the relationship between biofilm formation and antibiotic resistance and adhesion genes in Staphylococcus aureus strains isolated from raw cow milk in Shahrekord, Iran. A total of 90 samples of raw cow's milk were collected. Presumptive S. aureus strains were obtained using Baird-Parker plates after enrichment in tryptone soy broth, and final colonies were selected from brain heart infusion. Additional tests such as coagulase were done, and the identification was confirmed by the detection of the aroA gene. Biofilm producing strains were screened using a spectrophotometry method applied to microplates. Crystal violet staining was used to quantify the formation of biofilm. An antibiotic susceptibility test was performed using the Kirby-Bauer disc diffusion method. PCR was used to detect several biofilm and antibiotics resistance related genes. The chi-square test and Fisher's exact test were used to establish a statistically significant relationship between biofilm reaction and antibiotic resistance (p value <0.05). Results show a moderate (38.88%) recovery rate of S. aureus in milk and 65.71% of the isolates were strong biofilm producers. Antibiotic susceptibility tests show an alarming rate of resistance to beta-lactam antibiotics, especially penicillin (100%), ampicillin (91.42%), and oxacillin (71.42%). This finding correlates with antibiotic resistance gene detection, in which the gene blaZ was most found (71.42%), followed by mecA and Aac-D (42.85%). Detection of biofilm-related genes shows that all the genes targeted were found among S. aureus isolates. Statistical tests show a significant correlation between biofilm production and antibiotic resistance in S. aureus. This study revealed that there is a significant correlation between biofilm production and antibiotic resistance in S. aureus isolated from raw milk. These results highlight the need for regular surveillance of the occurrence of S. aureus strains in milk and milk products in Iran.

Emergence of Cfr-Mediated Linezolid Resistance among Livestock-Associated Methicillin-Resistant Staphylococcus aureus (LA-MRSA) from Healthy Pigs in Portugal.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 is mainly found in Europe and North America, colonizing the nasal cavity of pigs. This study characterized the MRSA isolates recovered from pig nasal swabs (n = 171) by evaluating the antimicrobial susceptibility profile by broth microdilution and characterizing the genetic lineages by spa-typing. Three linezolid-resistant isolates were subjected to Whole-Genome Sequencing (WGS). All strains harbored the mecA gene and were resistant to tetracycline and susceptible to vancomycin. A high frequency of multidrug resistance (97.6%) was evidenced, with 55 different multidrug resistance profiles identified. The MRSA strains were found to belong to 17 spa-types, three being novel. The linezolid-resistant strains appeared to belong to the ST398 type, spa-type t011, and SCCmec_type_Vc and to harbor the cfr, fexA, blaZ, mecA, tetM, and tetK genes. The cfr gene was predicted to be carried in the plasmid, flanked by ISSau9 and the transposon TnpR. MRSA from Portuguese fattening pigs present a high diversity of genetic lineages. The presence of cfr-positive LA-MRSA may represent a risk of transmission to humans, mainly to those in contact with livestock.

Occurrence and Characteristics of Staphylococcus aureus Strains along the Production Chain of Raw Milk Cheeses in Poland.

Cheeses produced from unpasteurized milk by traditional production methods may contain many groups of microorganisms, including Staphylococcus aureus. The aim of this study was to determine the occurrence of S. aureus in the artisanal cheese production chain from unpasteurized milk. We investigated the prevalence of S. aureus strains isolated from various stages of artisanal cheese of unpasteurized milk production from farms in the northeastern and southern parts of Poland and characterized them. Characterization included antimicrobial susceptibility by microbroth dilution and biofilm formation by in vitro assay. Among all strains, the presence of enterotoxigenic genes and genes involved with biofilm formation and antibiotic resistance were screened by PCR-based methods. A total of 180 samples were examined. A high percentage of strains were resistant to penicillin (54/58.1%) and tobramycin (32/34.4%). Some tested isolates also showed resistance to the macrolide class of antibiotics: azithromycin, clarithromycin, and erythromycin at 17/18.3%, 15/16.1%, and 21/22.6%, respectively. Among tested isolates, we also found phenotypic resistance to oxacillin (9/9.7%) and cefoxitin (12/12.9%). The blaZ gene encoding penicillin resistance was the most common gene encoding antibiotic resistance among the tested strains. All isolates showing phenotypic resistance to cefoxitin possessed the mecA gene. The study also evaluated the prevalence of biofilm-associated genes, with eno the most frequently associated gene. Eighty-nine out of 93 S. aureus isolates (95.7%) possessed at least one enterotoxin-encoding gene. The results of this study showed that production of raw milk cheeses may be a source of antibiotic resistance and virulent S. aureus. Our results suggest that artisanal cheese producers should better control production hygiene.

Prevalence of a cefazolin inoculum effect associated with blaZ gene types, and clinical outcomes among methicillin-susceptible Staphylococcus aureus blood isolates of patients with infective endocarditis

ObjectivesA proportion of blaZ gene-positive methicillin-susceptible Staphylococcus aureus (MSSA) strains exhibits the cefazolin inoculum effect (CInE). Its clinical impact remains uncertain but could compromise the use of cefazolin in high-burden infections. To date, no study has been conducted in France or in Europe. We aimed to assess the prevalence of CInE and its association with blaZ beta-lactamase and S. aureus protein A (spa) types, and to assess the clinical outcomes in cefazolin-treated patients for infective endocarditis whose strain exhibited a CInE. MethodsThis was a French single-center retrospective study of 51 MSSA strains from patients of the Nantes endocarditis prospective cohort, conducted between 2013 and 2018. ResultsCefazolin MIC50 at high inoculum was 2 mg/L (IQR 1–2). CInE was found in 17.6 % of tested strains. Among blaZ-positive strains (n = 44), type A beta-lactamase was predominant (n = 25, 57 %). Thirty-seven S. aureus protein A (spa) types were found. No statistical association was shown between blaZ or spa types and CInE. CInE was neither associated with a higher rate of persistent bacteremia (25 % vs 56.3 %, p = 0.58) nor with clinical failure in patients treated with cefazolin, in comparison to patients with no CInE strain (25 % vs 56.3 %, p = 0.58). ConclusionThe cefazolin inoculum effect was found in a substantial number of Staphylococcus aureus strains; however, minimum inhibitory concentrations remained globally low. CInE was not associated with a higher proportion of clinical failure on treatment.