Study of virulence factors in coagulase-negative staphylococci isolated from newborns

Coagulase-negative staphylococci (CNS) have been identified as the etiological agent in various infections and are currently the microorganisms most frequently isolated in nosocomial infections. However, little is known about the virulence factors produced by CNS that contribute to the pathogenesis of infections caused by these microorganisms. The study of CNS isolated from infectious processes of newborns hospitalized in the Neonatal Unit of the Hospital of the Botucatu Medical School, UNESP, indicated Staphylococcus epidermidis as the most frequently isolated species (77.8%), which was also associated with clinically significant situations. The analysis of virulence factors revealed the production of slime in 20 (17.1%) of all CNS samples isolated and the synthesis of a broad spectrum of enzymes and toxins, including hemolysins (19.6%), lipase (17.1%), lecithinase (3.4%), DNAse (15.4%), thermonuclease (7.7%), and enterotoxin A, B or C (37.6%). Taking into consideration that the etiological importance of CNS has often been neglected, the present investigation confirmed that these microorganisms should not be ignored or classified as mere contaminants.     

Susceptibility to selected coagulase-negative chemotherapeutic agents of methicillin resistant staphylococci isolated from clinical materials in 1997-1998

The study was aimed at assessment of the sensitivity of methicillin-resistant coagulase-negative staphylococci isolated from clinical material in 1997/1998 to selected chemotherapeutic agents. The investigated material comprised 96 methicillin-resistant coagulase-negative staphylococci from hospital and ambulatory infections isolated during the period from April 1997 to May 1998. Species affiliation was determined by classical identification methods and commercial diagnostic tests for identification of staphylococci. Methicillin resistance was determined by agar disk-diffusion method and screening. Sensitivity to chemotherapeutics was determined by agar disk-diffusion method and agar dilution methods. All the investigated strains were sensitive to nitrofurantoin, furazolidone and vancomycin. To teicoplanin--the second glycopeptide antibiotic--84% strains were sensitive, whereas the percentages of resistant and moderately sensitive strains amounted to 5.2% and 10.4%, respectively. 85% and 82% of coagulase-negative staphylococcal strains were sensitive to fusidic acid and mupirocin. Considerable differences were noted with respect to sensitivity to aminoglycoside group antibiotics. About 35% of strains were sensitive to gentamicin, and 90% sensitive to netilmicin. Ca. 40% of coagulase-negative staphylococci were resistant both to cotrimoxazole and trimethoprim, which, in view of 98% resistance to the second component of cotrimoxazole, may be associated with the activity of only one of the components of the drug--trimethoprim.     

Proposed virulence factors among coagulase-negative staphylococci isolated from two healthy populations

Proposed virulence factors, including multiple antibiotic resistance and slime-mediated adherence, among coagulase-negative staphylococci colonizing healthy individuals from two different study populations were examined. Resistance to methicillin was more commonly seen than initially anticipated. In addition, adherence characteristics, as quantitated by a microtiter plate method, were very similar to those of strains of coagulase-negative staphylococci causing nosocomial infections.     

Sensitivity to vancomycin and teicoplanin of coagulase-negative staphylococci isolated from clinical specimens

The frequency of resistance and elevated resistance to teicoplanin and vancomycin among 689 strains of coagulase-negative staphylococci isolated in one year from clinical specimens was determined. Using ATB.STAPH test, a resistance was shown mainly among strains of S. epidermidis and S. haemolyticus. The elevated resistance to teicoplanin was much more frequently observed than to vancomycin. About 27% of isolated strains of S. haemolyticus and 6.8% of S. epidermidis were classified as resistant. Among other species only single strains were recognised as resistant: one strain of S. xylosus, one of S. cohni and one of S. intermedius. 94.7% of S. epidermidis and 100% of S. haemolyticus strains classified as resistant to teicoplanin in ATB showed MIC values 14 mg/l. Moreover it was shown that 26.3% of these strains of S. epidermidis and 33.3% of S. haemolyticus had MBC of teicoplanin values equal to or higher than 32 mg/l.     

Plasmid profile and antibiotic resistance in coagulase-negative staphylococci isolated from polluted water

Four different species of coagulase-negative staphylococci (CNS) were isolated from polluted waters in Fez, Morocco and found to be Staphylococcus simulans, Staph. lenticus, Staph. hyicus and Staph. xylosus . Eight isolates belonging to these four species were analysed for their plasmid content. Southern blot hybridizations were performed to define the resistance determinants of the plasmids harboured by these species. These determinants were found to be carried mainly by Class I staphylococcal plasmids (1–5 kb). A plasmid (4·3 kb) carrying a tetracycline resistance gene was present in five isolates from all identified species. Plasmids carrying a chloramphenicol resistance gene were more frequently encountered and found to be of different sizes. Plasmids carrying erythromycin, neomycin, and streptomycin resistance genes were less frequent and were the same size. The results indicate that the occurrence of multi-resistant CNS in polluted waters may constitute a reservoir for disseminating antibiotic-resistance into the community.     

Identification of coagulase-negative staphylococci from farm animals

The species identity of 661 strains of coagulase-negative staphylococci isolated from the skin and nares of cattle, pigs, poultry, goats and sheep was determined. They belonged either to the novobiocin-sensitive species Staphylococcus hyicus, Staph. simulans, Staph. epidermidis, Staph. haemolyticus and Staph. warneri or to the novobiocin-resistant species Staph. sciuri, Staph. lentus, Staph. xylosus, Staph. cohnii. Staph. saprophyticus and Staph. gallinarum ; twenty-one strains remained unidentified. The staphylococcal flora of the farm animals studied differed markedly from that associated with man; several species which do not occur in man were isolated and novobiocin-resistant strains, which occur infrequently in man, were present in large numbers in animals. Two simplified schemes for the identification of staphylococci from farm animals and man are presented.     

Identification of coagulase-negative staphylococci from farm animals

The species identify of 661 strains of coagulase-negative staphylococci isolated from the skin and nares of cattle, pigs, poultry, goats and sheep was determined. They belonged either to the novobiocin-sensitive species Staphylococcus hyicus, Staph. simulans, Staph. epidermidis, Staph. haemolyticus and Staph. warneri or to the novobiocin-resistant species Staph. sciuri, Staph. lentus, Staph. xylosus, Staph. cohnii, Staph. saprophyticus and Staph. gallinarum; twenty-one strains remained unidentified. The staphylococcal flora of the farm animals studied differed markedly from that associated with man; several species which do not occur in man were isolated and novobiocin-resistant strains, which occur infrequently in man, were present in large numbers in animals. Two simplified schemes for the identification of staphylococci from farm animals and man are presented.     

Identification and methicillin resistance of coagulase-negative staphylococci isolated from nasal cavity of healthy horses

The aim of this study was an analysis of the staphylococcal flora of the nasal cavity of 42 healthy horses from 4 farms, along with species identification of CoNS isolates and determination of resistance to 18 antimicrobial agents, particularly phenotypic and genotypic methicillin resistance. From the 81 swabs, 87 staphylococci were isolated. All isolates possessed the gap gene but the coa gene was not detected in any of these isolates. Using PCR-RFLP of the gap gene, 82.8% of CoNS were identified: S. equorum (14.9%), S. warneri (14.9%), S. sciuri (12.6%), S. vitulinus (12.6%), S. xylosus (11.5%), S. felis (5.7%), S. haemolyticus (3.4%), S. simulans (3.4%), S. capitis (1.1%), S. chromogenes (1.1%), and S. cohnii subsp. urealyticus (1.1%). To our knowledge, this was the first isolation of S. felis from a horse. The species identity of the remaining Staphylococcus spp. isolates (17.2%) could not be determined from the gap gene PCR-RFLP analysis and 16S rRNA gene sequencing data. Based on 16S-23S intergenic transcribed spacer PCR, 11 different ITS-PCR profiles were identified for the 87 analyzed isolates. Results of API Staph were consistent with molecular identification of 17 (19.5%) isolates. Resistance was detected to only 1 or 2 of the 18 antimicrobial agents tested in the 17.2% CoNS isolates, including 6.9% MRCoNS. The mecA gene was detected in each of the 5 (5.7%) phenotypically cefoxitin-resistant isolates and in 12 (13.8%) isolates susceptible to cefoxitin. In total, from 12 horses (28.6%), 17 (19.5%) MRCoNS were isolated. The highest percentage of MRCoNS was noted among S. sciuri isolates (100%).     

Antibiotic resistance in coagulase-negative staphylococci isolated from Cope''s gray treefrogs (Hyla chrysoscelis)

Microcin J25 (MccJ25) is a cyclic peptide of 21 unmodified amino acid residues produced by a fecal strain of Escherichia coli. It has previously been shown that the antibiotic activity of this peptide is mainly directed to Enterobacteriaceae, including several pathogenic E. coli, Salmonella and Shigella strains. In this paper we show that MccJ25 acts on the cytoplasmic membrane of Salmonella newport cells producing alteration of membrane permeability, and the subsequent gradient dissipation, that initiate the inhibition of process, such as oxygen consumption. These results, taken together with our in vitro observations [Rintoul et al. (2000) Biochim. Biophys. Acta 1509, 65-72], strongly suggest that the disruption of the cytoplasmic membrane gradient is closely related to the bactericidal activity of MccJ25 in S. newport.     

Occurrence of high-level mupirocin resistant coagulase-negative staphylococci in hospitals and their characterization

Occurrence of high-level mupirocin resistant coagulase-negative staphylococci in 5 hospitals in the region of Gdansk was determined. The study was carried out on 192 staphylococcal strains isolated from patients and medical staff. The mupirocin resistant strains were detected by 5 microgram mupirocin disc. The minimal inhibitory concentration (MIC) for mupirocin was estimated by E-tests. The mupirocin resistant strains were characterised by antibiotic sensitivity, including MIC for glycopeptides and oxacillin. Biotypes of resistant strains were also determined. Eight high-level mupirocin resistant strains (4.7%) were found. Only one strain expressed low-level resistance. All but one of high-level mupirocin resistant strains were resistant to methicillin. Six of them belonged to the S. epidermidis species but differences in the biotypes and antibiotic resistance patterns of these strains suggest they did not have a common origin.     

Coagulase-negative staphylococci strains resistant to oxacillin isolated from neonatal blood cultures

Coagulase-negative staphylococci (CoNS) are the microorganisms most frequently isolated from clinical samples and are commonly found in neonatal blood cultures. Oxacillin is an alternative treatment of choice for CoNS infections; however, resistance to oxacillin can have a substantial impact on healthcare by adversely affecting morbidity and mortality. The objective of this study was to detect and characterise oxacillin-resistant CoNS strains in blood cultures of newborns hospitalised at the neonatal ward of the University Hospital of the Faculty of Medicine of Botucatu. One hundred CoNS strains were isolated and the mecA gene was detected in 69 of the CoNS strains, including 73.2% of Staphylococcus epidermidis strains, 85.7% of Staphylococcus haemolyticus strains, 28.6% of Staphylococcus hominis strains and 50% of Staphylococcus lugdunensis strains. Among these oxacillin-resistant CoNS strains, staphylococcal cassette chromosome mec (SCCmec) type I was identified in 24.6%, type II in 4.3%, type III in 56.5% and type IV in 14.5% of the strains. The data revealed an increase in the percentage of CoNS strains isolated from blood cultures from 1991-2009. Furthermore, a predominant SCCmec profile of the oxacillin-resistant CoNS strains isolated from neonatal intensive care units was identified with a prevalence of SCCmec types found in hospital-acquired strains.     

Antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine mastitis between 2003 and 2008 in Korea

A total of 1,444 coagulase-negative staphylococci (CNS) isolates from bovine mastitic milk samples collected during 2003-2008 in Korea were identified to the species level. Of 14 species identified, S. simulans, S. haemolyticus, and S. sciuri accounted for over 60% of the isolates. All the CNS isolates were tested for susceptibility to eight antimicrobials commonly used in dairy cattle. With a few exceptions, similar resistance patterns were observed among the CNS species: penicillin and ampicillin showed the lowest activity while amikacin, cephalothin, and gentamycin were highly effective. About 39% (557/1,444) of the CNS isolates were pan-susceptible, while 12% (175/1,444) showed resistance to four or more antimicrobials tested.     

Integron-bearing methicillin-resistant coagulase-negative staphylococci in South China, 2001–2004

A total of 53 methicillin-resistant coagulase-negative staphylococci strains isolated in a hospital in Guangzhou, China, were analyzed to detect class 1 integrons and SCC mec typing. Thirty strains had the class 1 integrase ( intI1 ) gene and 26 strains possessed the 3' conserved region of qacE Δ 1 - sul1 . Four different types of gene cassette arrays were found and a highly prevalent array of dfrA12-orfF-aadA2 gene cassettes was observed. Thirty class 1 integron-positive coagulase-negative staphylococci strains were subjected to Southern hybridization analysis; the result showed that class 1 integrons were located on chromosome, not plasmid. According to the results of SCC mec typing for 30 integron-bearing MRCNS strains, five, 15 and five strains belonged to type I, II and III SCC mec , respectively, and five strains were untypeable. For 23 non-integron-bearing methicillin-resistant coagulase-negative staphylococci strains, four, nine and seven strains belonged to type I, II and III SCC mec , respectively, and three strains were untypeable. None of the strains belonged to type IV or V. Twenty-three coagulase-negative staphylococci isolates of three Staphylococcal species that contained the dfrA12-orfF-aadA2 gene cassette array were phylogenetically unrelated to each other by randomly amplified polymorphic DNA, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer.     

Integron-bearing methicillin-resistant coagulase-negative staphylococci in South China, 2001-2004.

A total of 53 methicillin-resistant coagulase-negative staphylococci strains isolated in a hospital in Guangzhou, China, were analyzed to detect class 1 integrons and SCCmec typing. Thirty strains had the class 1 integrase (intI1) gene and 26 strains possessed the 3' conserved region of qacEDelta1-sul1. Four different types of gene cassette arrays were found and a highly prevalent array of dfrA12-orfF-aadA2 gene cassettes was observed. Thirty class 1 integron-positive coagulase-negative staphylococci strains were subjected to Southern hybridization analysis; the result showed that class 1 integrons were located on chromosome, not plasmid. According to the results of SCCmec typing for 30 integron-bearing MRCNS strains, five, 15 and five strains belonged to type I, II and III SCCmec, respectively, and five strains were untypeable. For 23 non-integron-bearing methicillin-resistant coagulase-negative staphylococci strains, four, nine and seven strains belonged to type I, II and III SCCmec, respectively, and three strains were untypeable. None of the strains belonged to type IV or V. Twenty-three coagulase-negative staphylococci isolates of three Staphylococcal species that contained the dfrA12-orfF-aadA2 gene cassette array were phylogenetically unrelated to each other by randomly amplified polymorphic DNA, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer.     

Production and novel quantification of haemolysins produced by coagulase-negative staphylococci isolated from subclinical mastitis in sheep

Coagulase-negative staphylococci producing cell-damaging toxins were isolated from the milk of sheep with subclinical mastitis. The haemolytic activity of Coagulase-negative staphylococcal strains was assessed on solid and liquid culture media. More than 61% and 76% of the tested strains on solid media produced evidence of alpha- and delta- haemolysins and more than 78% produced synergistic haemolysis. However almost all isolates producing haemolysin in liquid culture media produced only very few units of haemolysin compared to the positive control of five Coagulase-positive strains of staphylococci. It was concluded that solid media are better for classifying Coagulase-negative staphylococci as producers or not of haemolysins, and liquid media for measuring the size of this activity within the first few hours of intramammary infection.     

凝固酶阴性葡萄球菌耐药基因和粘附基因检测及耐药性分析

目的了解凝固酶阴性葡萄球菌（CoNS）耐药基因和粘附基因的分布及其耐药性情况。方法收集45株凝固酶阴性葡萄球菌临床分离株,采用PCR检测其耐药基因mecA和粘附基因c弘知如B,并分析其耐药性。结果（1）凝固酶阴性葡萄球菌中mecA、cva、fn6pB的阳性率依次为86．7％（39／45）、11．1％（5／45）、71．1％（32／45）。（2）凝固酶阴性葡萄球菌中耐甲氧西林凝固酶阴性葡萄球菌（MRCoNS）的检出率为88．9％,对β-内酰胺类抗生素的耐药率都在80％以上,对利福平、左氧氟沙星、林可霉素的耐药率较低,未发现对万古霉素、利奈唑胺耐药菌株。结论CoNS中MRCoNS检出率很高,对常用抗生素耐药率高,且有一定的粘附能力,临床上应该根据药敏试验结果慎重及合理的使用抗生素。     

Phagocytosis of chloramine B sensitive and resistant staphylococci isolated from healthy persons and patients

Phagocytic reaction with respect to antibiotic and chloramine B sensitive and resistant staphylococci isolated from healthy persons and patients, air and stock of medical institutions was studied on albino mice. It was shown that the staphylococcal isolates included strains simultaneously sensitive to antibiotics and chloramine, sensitive to antibiotics and resistant to chloramine, resistant to antibiotics and sensitive to chloramine and simultaneously resistant to antibiotics and chloramine. Activity, intensity and completeness of phagocytosis by leucocytes from mouse abdominal cavity exudates with respect to the staphylococcal strains sensitive to antibiotics and resistant to chloramine, resistant to antibiotics and sensitive to chloramine and simultaneously resistant to antibiotics and chloramine were lower than the values of the phagocytic reaction with respect to the isolates simultaneously sensitive to antibiotics and chloramine. This suggested that not only antibiotic resistance of microbes but also their resistance to disinfectants could be referred to complicating factors of hospital infections.