Staphylococcal Strains Research Articles

Association of Staphylococcal Populations on Teatcups of Milking Parlours with Vaccination against Staphylococcal Mastitis in Sheep and Goat Farms.

There is a paucity of information regarding staphylococcal populations on teatcups of milking parlours in sheep and goat farms. The objectives were to describe the populations of staphylococci on teatcups in milking parlours in sheep or goat farms in two field investigations throughout Greece and to potentially associate the findings with the use of anti-staphylococcal mastitis vaccinations in the farms visited during the two investigations. In a cross-sectional (255 sheep and 66 goat farms across Greece) and a longitudinal (12 sheep farms, four samplings, throughout lactation) study, swab samples were collected from 1418 teatcups (upper and lower part) for staphylococcal recovery, identification and assessment of biofilm-formation. A total of 328 contaminated teatcups (23.1%) were found in 105 sheep (41.2%) and 35 goat (53.0%) farms. Staphylococci were more frequently recovered from the upper than the lower part of teatcups: 269 versus 139 teatcups, respectively. After identification, 253 staphylococcal isolates were found: Staphylococcus aureus, Staphylococcus equorum, Staphylococcus lentus, and Staphylococcus capitis predominated. Of these isolates, 87.4% were biofilm-forming. The proportion of contaminated teatcups was smaller in farms where vaccination against anti-staphylococcal mastitis in general or vaccination specifically against mastitis caused specifically by biofilm-forming staphylococcal strains was applied, 19.7% or 10.9%, respectively, versus 25.5% in farms without vaccination. In the longitudinal study, contaminated teatcups were identified in 28 (58.3%) sampling occasions, with staphylococci being recovered more frequently from their upper part. The same species as in the cross-sectional study predominated. Of these isolates, 61.9% were biofilm-forming. In farms where vaccination against mastitis caused specifically by biofilm-forming staphylococcal strains was applied, the proportion of contaminated teatcups was smaller: 20.4% versus 48.3% in farms without vaccination. There were no differences in proportions of contaminated teatcups between sampling occasions. In conclusion, the great majority of staphylococci recovered from teatcups of milking parlours in sheep and goat farms included biofilm-forming isolates. Reduced staphylococcal isolation was noted in farms where anti-staphylococcal vaccination was performed; this was possibly the effect of reduced excretion of staphylococci in the milk of vaccinated animals.

First Case of Staphylococci Carrying Linezolid Resistance Genes from Laryngological Infections in Poland.

Linezolid is currently used to treat infections caused by multidrug-resistant Gram-positive cocci. Both linezolid-resistant S. aureus (LRSA) and coagulase-negative staphylococci (CoNS) strains have been collected worldwide. Two isolates carrying linezolid resistance genes were recovered from laryngological patients and characterized by determining their antimicrobial resistance patterns and using molecular methods such as spa typing, MLST, SCCmec typing, detection of virulence genes and ica operon expression, and analysis of antimicrobial resistance determinants. Both isolates were multidrug resistant, including resistance to methicillin. The S. aureus strain was identified as ST-398/t4474/SCCmec IVe, harboring adhesin, hemolysin genes, and the ica operon. The S. haemolyticus strain was identified as ST-42/mecA-positive and harbored hemolysin genes. Linezolid resistance in S. aureus strain was associated with the mutations in the ribosomal proteins L3 and L4, and in S. haemolyticus, resistance was associated with the presence of cfr gene. Moreover, S. aureus strain harbored optrA and poxtA genes. We identified the first case of staphylococci carrying linezolid resistance genes from patients with chronic sinusitis in Poland. Since both S. aureus and CoNS are the most common etiological factors in laryngological infections, monitoring of such infections combined with surveillance and infection prevention programs is important to decrease the number of linezolid-resistant staphylococcal strains.

Monitoring Methicillin-Resistant Staphylococcus Strains in the Moscow Medical and Surgical Center using Molecular-Biological Methods

Relevance. Staphylococci are one of the most common pathogens in the etiological structure of nosocomial infections. Methicillinresistant staphylococci are resistant to the main groups of antibiotics and are one of bacteria that pose the greatest threat to human health according to the World Health Organization. The introduction of modern molecular biological methods in the monitoring of methicillin-resistant staphylococci is necessary for the rapid and accurate identification of microorganisms, monitoring the epidemiological situation and studying the need for anti-epidemic measures. Aims. Аnalysis of the results of monitoring methicillinresistant staphylococcus strains using molecular biological methods in a multidisciplinary hospital in Moscow for one year. Materials and methods. Single-center observational study with a one-year follow-up period (December 2016 to December 2017). The research included a molecular-biological analysis of biological material from 240 patients with signs of infection and washings samples (n=250) from the objects of the hospital environment. The whole genome sequencing was carried out for 24 samples, isolated from patients with different forms of staphylococcal infection and washings from a hospital environment. DNA detection of methicillin-resistant strains was performed using the «AmpliSens®MRSA-screen-titer-FL» reagent kit. Sequencing was performed on an Illumina HiSeq1500 instrument using the IlluminaHiSeq PE RapidClusterKit v2 and IlluminaHiSeqRapid SBS Kit v2. Results. DNA of methicillin-resistant staphylococci were detected in 6.3% of blood samples from patients with signs of infection and in 42.4% of washings from the objects of the hospital environment. The results of PCR of washing samples showed that DNA of methicillin-resistant coagulase-negative staphylococci were detected much more often than MRSA (p <0.001). DNA of methicillin-resistant staphylococci were detected more often in the intensive care and intensive care units compared with the hematology and surgical departments (p <0.001). The examine samples of Staphylococcus aureus belonged to 6 different sequence types (ST-5, ST-7, ST-8, ST-22, ST-30 and ST-5555) and 8 spatypes (t008, t021, t091, t1062, t12437, t1544, t223, t4573). As a result of monitoring, an isolate with a new allelic profile was found. Today it has been assigned the number ST5555. The predominant type of staphylococcal mec cassette was the type IV SCCmec cassette. Conclusion. Due to the widespread distribution of methicillin-resistant strains and the identification of epidemiologically significant genetic lines of staphylococci, it is necessary to conduct regular monitoring, take measures to limit the spread of such strains and introduce modern molecular biological methods for quick and accurate identification.

Biofilm-Formation Ability and the Presence of Adhesion Genes in Coagulase-Negative Staphylococci Isolates from Chicken Broilers.

Simple SummaryBacteria of the genus Staphylococcus are universally present on the mucous membranes and skin of warm-blooded animals. They are divided into two groups on the basis of their ability to clot blood plasma: the coagulase-positive (CoPS) and coagulase-negative staphylococci (CoNS). Some species can cause opportunistic infections in poultry. Identification and characterization of strains of the genus Staphylococcus isolated from farm animals are crucial in epidemiological research and for developing effective methods to treat infections and food poisoning induced by these bacteria. The main virulence factor of coagulase-negative staphylococci is considered to be their ability to form complex biofilm structures on the surfaces of damaged tissues. Biofilms increase the invasive properties of CoNS and their ability to cause infection. The purpose of this study was to determine the biofilm-forming potential of coagulase-negative Staphylococcus strains isolated from poultry. The frequency of selected genes potentially playing a role in the biofilm formation process was also determined. The results of the study indicate that the majority (79.3%) of CoNS isolated from broiler chickens in this study were capable of producing a biofilm.The aim of the study was to analyze the biofilm-production capacity of 87 coagulase-negative Staphylococcus strains (CoNS) isolated from broiler chickens and to determine the occurrence of biofilm-associated genes. The biofilm production capacity of staphylococci was assessed using the microtiter plate method (MTP), and the frequency of genes was determined by PCR. The ability to form a biofilm in vitro was shown in 79.3% of examined strains. Strong biofilm capacity was demonstrated in 26.4% of strains, moderate capacity in 25.3%, weak capacity in 27.6%, and a complete lack of biofilm production capacity in 20.7% of strains. The icaAB gene responsible for the production of extracellular polysaccharide adhesins was detected in 6.9% of strains. The other four genes, i.e., bap (encoding biofilm-associated protein), atlE (encoding cell surface protein exhibiting vitronectin-binding activity), fbe (encoding fibrinogen-binding protein), and eno (encoding laminin-binding protein) were detected in 5.7%, 19.5%, 8%, and 70.1% of strains, respectively. Demonstration of genes that play a role in bacterial biofilm formation may serve as a genetic basis to distinguish between symbiotic and potentially invasive coagulase-negative staphylococcal strains.

The antimicrobial activity and biocompatibility of a controlled gentamicin-releasing single-layer sol-gel coating on hydroxyapatite-coated titanium.

AimsThe aim of this study was to develop a single-layer hybrid organic-inorganic sol-gel coating that is capable of a controlled antibiotic release for cementless hydroxyapatite (HA)-coated titanium orthopaedic prostheses.MethodsCoatings containing gentamicin at a concentration of 1.25% weight/volume (wt/vol), similar to that found in commercially available antibiotic-loaded bone cement, were prepared and tested in the laboratory for: kinetics of antibiotic release; activity against planktonic and biofilm bacterial cultures; biocompatibility with cultured mammalian cells; and physical bonding to the material (n = 3 in all tests). The sol-gel coatings and controls were then tested in vivo in a small animal healing model (four materials tested; n = 6 per material), and applied to the surface of commercially pure HA-coated titanium rods.ResultsThe coating released gentamicin at > 10 × minimum inhibitory concentration (MIC) for sensitive staphylococcal strains within one hour thereby potentially giving effective prophylaxis for arthroplasty surgery, and showed > 99% elution of the antibiotic within the coating after 48 hours. There was total eradication of both planktonic bacteria and established bacterial biofilms of a panel of clinically relevant staphylococci. Mesenchymal stem cells adhered to the coated surfaces and differentiated towards osteoblasts, depositing calcium and expressing the bone marker protein, osteopontin. In the in vivo small animal bone healing model, the antibiotic sol-gel coated titanium (Ti)/HA rod led to osseointegration equivalent to that of the conventional HA-coated surface.ConclusionIn this study we report a new sol-gel technology that can release gentamicin from a bioceramic-coated cementless arthroplasty material. In vitro, local gentamicin levels are in excess of what can be achieved by antibiotic-loaded bone cement. In vivo, bone healing in an animal model is not impaired. This, thus, represents a biomaterial modification that may have the potential to protect at-risk patients from implant-related deep infection.Cite this article: Bone Joint J 2021;103-B(3):522–529.

Antibiotic Resistance of Staphylococcal Strains Isolated from Patients with Purulent-Septic Infections and Creation of an Anti-Staphylococcal Phage Preparation

Antibiotic Resistance of Staphylococcal Strains Isolated from Patients with Purulent-Septic Infections and Creation of an Anti-Staphylococcal Phage Preparation

Protein A Modulates Neutrophil and Keratinocyte Signaling and Survival in Response to Staphylococcus aureus.

The type 1 TNF-α receptor (TNFR1) has a central role in initiating both pro-inflammatory and pro-apoptotic signaling cascades in neutrophils. Considering that TNFR1 signals Staphylococcus aureus protein A (SpA), the aim of this study was to explore the interaction of this bacterial surface protein with neutrophils and keratinocytes to underscore the signaling pathways that may determine the fate of these innate immune cells in the infected tissue during staphylococcal skin infections. Using human neutrophils cultured in vitro and isogenic staphylococcal strains expressing or not protein A, we demonstrated that SpA is a potent inducer of IL-8 in neutrophils and that the induction of this chemokine is dependent on the SpA-TNFR1 interaction and p38 activation. In addition to IL-8, protein A induced the expression of TNF-α and MIP-1α highlighting the importance of SpA in the amplification of the inflammatory response. Protein A contributed to reduce neutrophil mortality prolonging their lifespan upon the encounter with S. aureus. Signaling initiated by SpA modulated the type of neutrophil cell death in vitro and during skin and soft tissue infections (SSTI) in vivo triggering the apoptotic pathway instead of necrosis. Moreover, SpA induced pro-inflammatory cytokines in keratinocytes, modulating their survival in vitro and preventing the exacerbated necrosis and ulceration of the epithelium during SSTI in vivo. Taken together, these results highlight the importance of the inflammatory signaling induced by protein A in neutrophils and skin epithelial cells. The ability of protein A to modulate the neutrophil/epithelial cell death program in the skin is of clinical relevance considering that lysis of neutrophils and epithelial cells will promote an intense inflammatory response and contribute to tissue damage, a non-desirable feature of complicated SSTI.

Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay.

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases worldwide, resulting from the ingestion of staphylococcal enterotoxins (SEs), primarily SE type A (SEA), which is produced in food by enterotoxigenic strains of staphylococci, mainly S. aureus. Since newly identified SEs have been shown to have emetic properties and the genes encoding them have been found in food involved in poisoning outbreaks, it is necessary to have reliable tools to prove the presence of the toxins themselves, to clarify the role played by these non-classical SEs, and to precisely document SFP outbreaks. We have produced and characterized monoclonal antibodies directed specifically against SE type G, H or I (SEG, SEH or SEI respectively) or SEA. With these antibodies, we have developed, for each of these four targets, highly sensitive, specific, and reliable 3-h sandwich enzyme immunoassays that we evaluated for their suitability for SE detection in different matrices (bacterial cultures of S. aureus, contaminated food, human samples) for different purposes (strain characterization, food safety, biological threat detection, diagnosis). We also initiated and described for the first time the development of monoplex and quintuplex (SEA, SE type B (SEB), SEG, SEH, and SEI) lateral flow immunoassays for these new staphylococcal enterotoxins. The detection limits in buffer were under 10 pg/mL (0.4 pM) by enzyme immunoassays and at least 300 pg/mL (11 pM) by immunochromatography for all target toxins with no cross-reactivity observed. Spiking studies and/or bacterial supernatant analysis demonstrated the applicability of the developed methods, which could become reliable detection tools for the routine investigation of SEG, SEH, and SEI.

Phenotypic and genotypic virulence features of staphylococcal strains isolated from difficult-to-treat skin and soft tissue infections

Chronic infections represent an important burden on the healthcare system and have a significant impact on the patients’ quality of life. While Staphylococcus spp. are commensal bacteria, they can become pathogenic, leading to various types of infections. In this study we aimed to characterize the virulence profiles of staphylococcal strains involved in difficult-to-treat skin and soft tissue infections, from both phenotypic and genotypic points of view. Phenotypic ability of the strains to secrete soluble virulence factors was assessed by a culturing dependent assay and their capacity to develop biofilms on inert substrate was screened by an adapted crystal violet microtiter method. We also tested the presence of several virulence genes by PCR. Most of the studied strains were isolated from purulent secretions of acne lesions and frequently secreted two or three soluble virulence factors. Most frequently secreted soluble virulence factors were caseinase (89%), lipase (71%) and lecithinase (67%). Almost half of the strains produced a well-represented biofilm. The molecular characterization showed the presence of the genes cna, hlg, clfA, and clfB. Staphylococcal strains that produce difficult-to-treat skin and soft tissue infections seem to be characterized by an enhanced ability to produce different soluble virulence factors and to develop biofilms in vitro. Further studies need to be developed in other Staphylococcus spp. infections in order to confirm this hypothesis.

IDENTIFICATION OF STAPHYLOCOCCUS SPP. ISOLATED FROM FOOD BY TWO METHODS

One hundred and ninety-three staphylococcal strains were isolated from samples of alaska pollock meat (Theragra chalcogramma), atlantic mackerel meat (Scomber scombrus), atlantic herring meat (Clupea harengus), and femoral muscle samples of the wild pheasants (Phasianus colchicus), wild rabbits (Oryctolagus cuniculus) and bryndza cheese. Phenotypic manifestations typical of individual staphylococcal species were detected in these isolates. Species identification was also performed by matrix-assisted-laser-desorption-ionization-mass-spectrometry on the basis of which 5 species of staphylococci were determined: S. aureus, S. epidermidis, S. capitis, S. haemolyticus, S. warneri. Subsequently, the presence of the 16S rDNA gene was confirmed by PCR assay; a specific sequence for S. aureus species; putative transcriptional regulator gene serp0107, a specific sequence of the sodA gene was used to identify S. epidermidis and to identify S. warneri, S. haemolyticus, S. capitis species. When comparing the results of identification by PCR assay and matrix-assisted-laser-desorption-ionization-mass-spectrometry, the identification at the genus level was identical in all cases (100%). However, the discrepancies in results were confirmed at the species level, where congruence for identification of isolates by PCR assay and matrix-assisted-laser-desorption-ionization-mass-spectrometry was only 80%. For these isolates, the score value ranged from 1.953 to 2.564.

Prevalence and Antibiotic Resistance Pattern of Staphylococcus aureus isolates from Clinical samples at a Tertiary Care Hospital, North India

Background: Staphylococcus aureus (S. aureus) is a well recognized nosocomial and community acquired pathogen which is implicated in causing a wide spectrum of superficial, deep pyogenic infections and toxin mediated illnesses. Localized infections sometimes progress to systemic infections, while ‘spontaneous’ bacteraemia also occur without an evident septic focus, particularly in chronic debilitated immunocompromised patients. Emergence of drug resistance to penicillins and penicillinase-resistant penicillins (i.e., oxacillin and methicillin) is a globally recognized problem. Methicillin Resistant Staphylococcus aureus isolates are also important with respect to the range of nosocomial infections it causes, leading to an increase in the hospital expenditure and mortality or morbidity rate. The increased prevalence of such resistant strains has narrowed down the list of available therapeutic options. Therefore, information regarding the prevalence of S. aureus infections in a health care setting and determining its current antibiotic resistance profile becomes crucial in selecting appropriate treatment regime. Therefore, the current study was done in the department of Microbiology to determine the prevalence of S. aureus infections and the antibiotic resistance pattern of S. aureus isolates from various clinical specimens at our tertiary care hospital in North India. Materials and Methods: A one year prospective study was carried out in the Department of Microbiology, at a tertiary care hospital in North India where non-duplicate strains of S. aureus isolated from various clinical specimens received in the Microbiology laboratory were studied. All S. aureus isolates were subjected to Antibiotic Susceptibility Testing using Kirby Bauer’s disk diffusion method on Mueller Hinton Agar plates in accordance to CLSI guidelines. The antibiotics tested included Penicillin (10U), Amoxicillin-clavulanic acid (20/10μg), Sulphamethoxazole-trimethoprim (1.25/23.75μg), Ciprofloxacin (5μg), Erythromycin (15μg), Clindamycin (2μg), Vancomycin (30μg), Teicoplanin (30μg) and Linezolid (30μg). Results: A total of 23,699 clinical specimens were processed in the laboratory while conducting this study, from which 1233 clinical isolates of S. aureus were identified and processed further. Among all clinical specimens, pyogenic samples (63.1%) yielded maximum number of S. aureus strains followed by blood samples (29.9%) and urine samples (4.8%). S. aureus infection was more evident in hospitalized 71.2% patients than in OPD patients 28.8%. Seasonal variation was also seen in isolation of S. aureus, with a higher percentage of isolates obtained during summer season than during winter season. On antibiotic susceptibility testing, 49.6% strains were Methicillin Resistant. Majority of the isolates were found resistant to Penicillin (92.1%), followed by Erythromycin (59%). Almost half of the total isolates were resistant to Sulphamethoxazole-Trimethoprim (49.3%) followed by Amoxicillin- Clavulanic acid (47.8%), Ciprofloxacin (43.4%) and Clindamycin (18.4%). Antibiotics to which all isolates showed 100% susceptibility included Vancomycin, Teicoplanin, Linezolid. Conclusion: Given the high prevalence of resistance to antibiotics seen in this study, effective treatment of infections caused by multidrug resistant Staphylococcal strains may become challenging. Drugs like Vancomycin, Teicoplanin and Linezolid promise to work as miracle drugs against the multidrug resistant MRSA strains but we need to warrant judicious use of these wonder drugs to conserve them for future use.

Antibacterial therapy of pyogenic liver abscesses

Purpose of the study. To study the features of the microbial landscape, to develop an algorithm for antibacterial therapy in patients with pyogenic liver abscesses. Prove the effectiveness of the developed algorithm in the treatment of such patients.
 Materials and methods. The results of treatment of 79 patients with pyogenic liver abscesses were analyzed. The mean age of patients was 48,4 ± 4,7 years, with men predominating (62,9%). The inclusion criterion was the presence of pyogenic liver abscesses, the exclusion criteria were cholangiogenic and specific abscesses. The main group consisted of 44 patients operated on minimally invasive technologies based on modern diagnostic methods. They received a comprehensive conservative therapy, which took into account antibacterial treatment according to the developed algorithm. The tactics of surgical treatment of 35 patients in the control group did not differ from the main one. The groups did not differ significantly in age, sex, comorbidity, severity of the disease and the results of microbiological examination.
 Results. The number of inoculations of microorganisms from the purulent center was dominated by monoinfection – 86,4–88,6% of cases, which was mostly aerobic. Most of them were facultative anaerobic bacteria of the genus Enterobacteriaceae, namely Kl. pneumoniae – 34,1–40,1%. Less than a quarter of patients (11,4–13,6%) isolated cultures are represented by microbial associations – aerobic-anaerobic and aerobicaerobic. However, aerobic-aerobic infection prevailed – 75,0–83,3%. MRSA strains of staphylococci were isolated in 5,7–6,8% of patients. Treatment began on the first day of hospitalization. Empirical treatment was immediately prescribed, followed by antibiotic replacement, if necessary, based on the results of microbiological examination and determination of the sensitivity of the isolated cultures. According to microbiological studies, in patients with AP most of the isolated microorganisms (71,4–90,9%) were sensitive to linezalide and taigecycline. These antibacterial drugs were reserve drugs and were prescribed in extremely severe cases in the absence of sensitivity to other drugs. The effectiveness of treatment was evaluated by clinical data and improvement of laboratory parameters (normalization of body temperature and leukocyte formula).
 Conclusion. Carrying out adequate combined antibiotic therapy for patients with AP according to the developed algorithms, along with surgery, allowed to reduce the recovery time of patients: for 2–3 days normalization of body temperature (t = 5,66176; P < 0,000001) and leukocyte formula (t = 8, 56860; P < 0,000001) patients of the main group in comparison with control patients. In turn, this contributed to a probable reduction in the length of stay of the patient in the hospital for 3 days (t = 3,95561; P = 0,000116).

Profiles of coagulase-positive and -negative staphylococci in retail pork: prevalence, antimicrobial resistance, enterotoxigenicity, and virulence factors.

ObjectiveThe present study aimed to investigate the occurrence and species of coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS) in retail pork meat samples collected during nationwide monitoring. The staphylococcal isolates were characterized for antimicrobial and zinc chloride resistance and enterotoxigenic potential.MethodsA total of 260 pre-packaged pork meat samples were collected from 35 retail markets in 8 provinces in Korea for isolation of staphylococci. Antimicrobial and zinc chloride resistance phenotypes, and genes associated with the resistance phenotypes were determined on the isolates. Furthermore, the presence and distribution of 19 staphylococcal enterotoxin (SE) genes and enterotoxin-like genes among the pork-associated staphylococci were determined by multiplex polymerase chain reaction-based assays using the specific primer sets.ResultsA total of 29 staphylococcal strains (29/260, 11.1%) were isolated from samples of retail pork meat, 24 (83%) of which were CoNS. The four CoNS species identified were S. saprophyticus (n = 16, 55%), S. sciuri (n = 3, 10%), S. warneri (n = 3, 10%), and S. epidermidis (n = 2, 7%). Among the 29 isolates, four methicillin-resistant CoNS (MR-CoNS; three S. sciuri and one S. epidermidis) and one methicillin-resistant CoPS (MR-CoPS; one S. aureus) were identified. In addition, a relatively high level of tetracycline (TET) resistance (52%) was confirmed in CoNS, along with a predominant distribution of tet(K). The most prevalent SEs were sep (45%), and sen (28%), which were carried by 81% of S. saprophyticus.ConclusionThese findings suggest that CoNS, especially S. saprophyticus strains, in raw pork meat could be a potential risk factor for staphylococcal food poisoning (SFP), and therefore, requires further investigation to elucidate the role of SEls in SFP and virulence of the pathogen. Our results also suggest that CoNS from raw pork meat may act as a source for transmission of antimicrobial resistance genes such as staphylococcal cassette chromosome mec and tet(K).

Comparative study for the efficacy of Sofinox cream against Staphylococcus aureus with Fucidin cream, T-Bact ointment and screening for resistant mutants.

Objective: To determine the Minimum Inhibition Concentration of Sofinox cream, Fucidin cream, T Bact ointment against Staphylococcus aureus and to screen for the development of resistant mutants and Whole Genome Sequencing(WGS) of the strains used and the strains showing raised MIC following serial passages. Material and Methods: An in vitro study was conducted to determine the Minimum Inhibition Concentration of Sofinox cream, Fucidin cream, T Bact ointment against seven strains of Staphylococcus aureus (ATCC 25923, ATCC 43300, ATCC 700699, 2 clinical isolates of methicillin-sensitive S. aureus (MSSA) and 2 methicillin-resistant S. aureus (MRSA). We also screened the same strains for the development of resistant mutants of Staphylococcus aureus by sub MIC exposure to Sofinox cream, Fucidin cream and T Bact ointment up to 70 serial passage and their WGS. Results: The MIC values of the three topical antibiotics ranged between 16 µg to 64 µg for all the strains of Staphylococcal strains tested. The MIC value did not change for three strains of Staphylococcus aureus and a one-fold rise in MIC value occurred for four strains of the Staphylococcus aureus strains for Sofinox cream after 70 passages at sub MIC concentration. WGS analysis of the strains with Sofinox cream treatment, it was noted that 150 number significant genetic variations to MSSA (ATCC 25923) and 84 number for MRSA (ATCC 700699) and no significant changes with other strains. For two of the strains the MIC value remained the same, three strains showed the one-fold rise and two strains showed a four-fold rise in MIC value for Fucidin cream after 70 passages at sub MIC concentration. Through the WGS analysis of the strains, it was noted that 185 significant genetic variations to one of the clinical isolates of MSSA followed by 102 to MRSA standard strain ATCC700699, the strains showing a one-fold rise in MIC showed 95, 85 and 76 number of genetic variations respectively. All the strains of Staphylococcus aureus showed a rise in MIC for T Bact which ranged from one to four-fold rise. WGS analysis of the strains with T Bact ointment treatment, it was noted the variations ranging from 85 to 158 with a one-fold rise to a four-fold rise in MIC value. Conclusions: In the present study it was observed that Sofinox cream had low resistance potential in vitro compared to Fucidin cream whereas T bact ointment had more resistance potential when exposed to sub MIC concentration of the cream/ointment. Even the WGS analysis showed variations correlating with MIC values. Hence there is a less likely chance of development of resistant mutants with the topical use of Sofinox cream even up to 8 weeks.

Prebiotic Combinations Effects on the Colonization of Staphylococcal Skin Strains.

Background: An unbalanced skin microbiota due to an increase in pathogenic vs. commensal bacteria can be efficiently tackled by using prebiotics. The aim of this work was to identify novel prebiotic combinations by exerting species-specific action between S. aureus and S. epidermidis strains. Methods: First, the antimicrobial/antibiofilm effect of Xylitol-XYL and Galacto-OligoSaccharides–GOS combined with each other at different concentrations (1, 2.5, 5%) against S. aureus and S. epidermidis clinical strains was evaluated in time. Second, the most species-specific concentration was used to combine XYL with Fructo-OligoSaccharides–FOS, IsoMalto-Oligosaccharides–IMO, ArabinoGaLactan–LAG, inulin, dextran. Experiments were performed by OD600 detection, biomass quantification and LIVE/DEAD staining. Results: 1% XYL + 1% GOS showed the best species-specific action with an immediate antibacterial/antibiofilm action against S. aureus strains (up to 34.54% ± 5.35/64.68% ± 4.77) without a relevant effect on S. epidermidis. Among the other prebiotic formulations, 1% XYL plus 1% FOS (up to 49.17% ± 21.46/37.59% ± 6.34) or 1% IMO (up to 41.28% ± 4.88/36.70% ± 10.03) or 1% LAG (up to 38.21% ± 5.31/83.06% ± 5.11) showed antimicrobial/antibiofilm effects similar to 1% XYL+1% GOS. For all tested formulations, a prevalent bacteriostatic effect in the planktonic phase and a general reduction of S. aureus biofilm formation without loss of viability were recorded. Conclusion: The combinations of 1% XYL with 1% GOS or 1% FOS or 1% IMO or 1% LAG may help to control the balance of skin microbiota, representing good candidates for topic formulations.

Prevalence of antimicrobial-resistant staphylococci in nares and affected sites of pet dogs with superficial pyoderma.

Currently, antimicrobial-resistant staphylococci, particularly methicillin-resistant Staphylococcus pseudintermedius (MRSP), are frequently isolated from canine superficial pyoderma in Japan. However, little is known regarding the nasal prevalence of MRSP in pet dogs. Here, we determined the prevalence of antimicrobial-resistant staphylococci in nares and affected sites of pet dogs with superficial pyoderma. Of the 125 nares and 108 affected sites of pet dogs with superficial pyoderma, 107 (13 species) and 110 (eight species) staphylococci strains, respectively, were isolated. The isolation rate of S. pseudintermedius from pyoderma sites (82/110 strains, 74.5%) was significantly higher than that from nares (57/107 strains, 53.3%) (P<0.01). Notably, the prevalence of MRSP (18/57 strains, 31.6%) in nares was equivalent to that in pyoderma sites (28/82 strains, 34.1%). Furthermore, the phenotypes and genotypes of antimicrobial resistance in MRSP strains from nares were similar to those from pyoderma sites. Our findings revealed that the prevalence of antimicrobial-resistant staphylococci in the nares of pet dogs with superficial pyoderma is the same level as that in affected sites. Therefore, considerable attention should be paid to the antimicrobial resistance of commensal staphylococci in companion animals.

Antimicrobial profile of inducible clindamycin resistant strains of staphylococcus species

Introduction: The appearance of continuous resistant to multiple drugs among Staphylococci is a global burden due to its ability to cause severe infections. The selective use of drugs is necessary to overwhelm the situation. Taking in account present study was carried out to rule out true susceptibility of clindamycin towards staphylococcus species and its antimicrobial profile for judicial use of the drugs. Material and Methods: All the clinical samples received in the Department of microbiology were screened for Staphylococci as per standard guidelines which were further subjected to Antimicrobial susceptibility testing and D-test to detect MLSb phenotypes. Results: A total of 421 Staphylococcus species, 359(85.3%) were Staphylococcus aureus and 62(14.8%) were Coagulase negative Staphylococci; among them 42(10%) were Staphylococcus epidermidis& 20(4.8%) were Staphylococcus saprophyticus. D-test for S.aureus shows that 173(48.2%) inducible Clindamycin resistant, 113(31.5%) strains were constitutive MLSb phenotypes and 58(16.2%) strains shown to have MSb phenotypes. Among CoNS; among Staphylococcus epidermidis and S.saprophyticus 9.5% & 5% were Inducible Clindamycin resistance, 38.1% & 85% were constitutive MLSb phenotypes and 28.6%& 10% were MSb phenotypes respectively. All the isolates were sensitive to Linezolid, Vancomycin and Ceftaroline. Conclusion: Inducible clindamycin resistant strains of Staphylococci found to be among half of the strains, indicating that true susceptibility of clindamycin should be rule out on routine basis for proper institution of the therapy. Keywords: Staphylococci, MLSb phenotypes, AST.

Efficacy of Daptomycin-Containing Regimen for Treatment of Staphylococcal or Enterococcal Vertebral Osteomyelitis: A Prospective Clinical Experience

Vertebral osteomyelitis (VO) is a compelling clinical entity for clinicians, because of its insidious and indolent course that makes diagnosis difficult. A concern is reported about the choice of antibiotic regimens, duration of therapy, and criteria to switch to oral therapy. We conducted a prospective observational study. All consecutive hospitalized patients with a confirmed diagnosis of VO caused by staphylococcal or enterococcal strains were analyzed. The primary endpoint was the analysis of clinical cure at the end of therapy. A propensity score for receiving therapy with daptomycin was added to the model. During the study period, 60 episodes of confirmed VO were observed. The main etiology of infection was methicillin-resistant Staphylococcus aureus (29%). Overall, clinical failure at end of therapy was reported in 11 (18.3%) patients. Logistic regression analysis, after propensity score, showed that >2 vertebrae involved (OR 2.4, CI95% 1.12–5.24, p = 0.002) and inadequate drainage of infection (OR 4.8, CI95% 2.45–8.51, p < 0.001) were independently associated with failure of therapy, while the use of a daptomycin-containing-regimen (OR 0.15, CI 95% 0.04–0.46, p < 0.001) with clinical cure. VO caused by staphylococcal or enterococcal strains is associated with an important rate of clinical failure. Daptomycin-containing regimen was strongly associated with clinical cure. Considering that over 70% of VO etiology is caused by Gram-positive strains but the etiology of infection is obtained in about 75% of cases, these data may help physicians to choose the appropriate antibiotic regimen.

In Vitro Evaluation of Gentamicin or Vancomycin Containing Bone Graft Substitute in the Prevention of Orthopedic Implant-Related Infections.

Antibiotic-loaded bone graft substitutes are attractive clinical options and have been used for years either for prophylaxis or therapy for periprosthetic and fracture-related infections. Calcium sulfate and hydroxyapatite can be combined in an injectable and moldable bone graft substitute that provides dead space management with local release of high concentrations of antibiotics in a one-stage approach. With the aim to test preventive strategies against bone infections, a commercial hydroxyapatite/calcium sulfate bone graft substitute containing either gentamicin or vancomycin was tested against Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, harboring different resistance determinants. The prevention of bacterial colonization and biofilm development by selected microorganisms was investigated along with the capability of the eluted antibiotics to select for antibiotic resistance. The addition of antibiotics drastically affected the ability of the selected strains to adhere to the tested compound. Furthermore, both the antibiotics eluted by the bone graft substitutes were able to negatively impair the biofilm maturation of all the staphylococcal strains. As expected, P. aeruginosa was significantly affected only by the gentamicin containing bone graft substitutes. Finally, the prolonged exposure to antibiotic-containing sulfate/hydroxyapatite discs did not lead to any stable or transient adaptations in either of the tested bacterial strains. No signs of the development of antibiotic resistance were found, which confirms the safety of this strategy for the prevention of infection in orthopedic surgery.

Structural and biochemical characteristics of two Staphylococcus epidermidis RNase J paralogs RNase J1 and RNase J2

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.