Occurrence of beta-lactamase type ESBL in rods of the Serratia species

Serratia spp. has been identified as an important opportunistic pathogen agent in nosocomial infections. The aim of the study was the determination of extended spectrum beta-lactamases (ESBL) occurrence among 78 of Serratia spp. strains isolated in 1996-1998 from clinical specimens obtained from patients of State Clinical Hospital in Bydgoszcz. Identification of Serratia spp. strains was performed in automatic ATB system with ID 32GN strips (bioMérieux). The strains with ESBL activity were detected by double-disc method according to Jarlier et al. (10) with small modifications. Clavulanic acid, tazobactam and sulbactam were used as the inhibitors of ESBLs. Drug-susceptibility was determined by disc-diffusion method according to NCCLS standards. Forty-five (57.7%) of the strains were ESBL (+). All of them belonged to S. marcescens species. The majority--91.1% of strains was derived from urine, 3 from wound and 1 from blood. The obtained results indicate the necessity of monitoring of ESBL-producing strains among gram-negative rods from clinical specimen. The aims of such a procedure are to control and to prevent their dissemination within hospital, as well as to avoid therapeutic failures.     

Antiseptic susceptibility and distribution of antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus

We examined the antiseptic susceptibilities and distribution of antiseptic-resistance genes qacA and smr in 98 isolates of methicillin-resistant Staphylococcus aureus obtained in 1992. Seventy-one strains were resistant to antiseptics. The qacA and smr genes were detected in 10 and 20 strains, respectively. The remaining 41 strains without qacA and smr were divided into two groups that exhibited low-level (n = 22) and high-level (n = 19) resistance to acriflavin. DNA cloning and sequencing suggested that norfloxacin-resistance gene norA was responsible for the high-level resistance to acriflavin. Our results indicated that four or more antiseptic-resistance genes exist in methicillin-resistant S. aureus and that antiseptic-resistant methicillin-resistant S. aureus strains without qacA and smr are widely spread in Japan.     

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.     

Drug resistance of 100 clinical strains of Enterococcus spp

The aim of this study was to evaluate the drug susceptibility of 100 Enterococcus spp. strains isolated from patients hospitalized in State Clinical Hospital No 1 in Warsaw. All strains were identified (API 20 STREP) and their susceptibility to antibiotics was tested (ATB STREP) in automatic ATB system. Additionally, PYRase activity, beta-lactamase production (in nitrocefin test), MICs for vancomycin and teicoplanin (E test), HLAR--high level aminoglycoside resistance and susceptibility to vancomycin, teicoplanin, piperacillin and piperacillin/tazobactam (disc diffusion method) were determined. E. faecalis ATCC 29212 was used as the control strain. Fifty E. faecalis, 45 E. faecium, 2 E. casseliflavus, 2 E. durans and 1 E. avium strain were cultured. All strains were PYRase-positive and beta-lactamase-negative. Ten isolates demonstrated intermediate susceptibility to vancomycin (6--E. faecalis and 4--E. faecium). One E. faecalis strain was intermediately susceptible to both glycopeptides. One E. casseliflavus strain showed low-level resistance to vancomycin, but this strain was susceptible to teicoplanin--phenotype Van C. HLAR strains were found among 31 E. faecalis and 40 E. faecium strains. 48 E. faecalis strains were susceptible to piperacillin and 49 to piperacillin/tazobactam. Whereas, 41 E. faecium were resistant to both these drugs. Thirty six per cent of isolates were resistant to penicillin and ampicillin, 73% to erythromycin, 87% to tetracycline, 89% to lincomycin and 56% to nitrofurantoin. Some discrepancies were noticed between the results of different methods applied for susceptibility testing--ATB system, E test and disc diffusion. These discrepancies concerned HLAR detection and susceptibility to glycopeptides determination. The best methods were: disc-diffusion for HLAR detection and E test for determination of resistance to vancomycin and teicoplanin. Increasing resistance to antimicrobial agents is observed in clinical Enterococcus spp. isolates cultured in our laboratory, especially in E. faecium strains. It is necessary to control the dissemination of multiresistant Enterococcus spp. strains in hospital wards.     

Co-trimoxazole resistance in methicillin-resistant staphylococci isolated from clinical material in 2001-2003

The aim of this study was to determine resistance to co-trimoxazole among 476 strains of methicillin-resistant Staphylococus aureus (MRSA) and 2137 strains of methicillin resistant coagulase-negative staphylococci (MRCNS) isolation during three years (2001-2003). Their susceptibility testing were performed by the disc-diffusion techniques according to recomendation of NCCLS (National Committe for Clinical Laboratory Standards). Co-trimoxazole resistance was demonstrated more frequently among MRCNS (52,8%, 51,4%, 63,9% in 2001, 2002, 2003), than among MRSA (23,4%, 19,5%, 16,8% adequately).     

Detection of the intercellular adhesion loci (ica) in clinicalStaphylococcus aureus strains responsible for hospital acquired auricular infection

In clinicalStaphylococcus aureus strains, the presence of theica genes, biofilm formation and susceptibility to antibiotics are considered important factors of virulence. In this study, 35 strains ofS. aureus, isolated from auricular infection, were investigated for slime production using Congo red agar (CRA) method, antibiotic susceptibility, presence ofmecA gene, and presence oficaA andicaD gene. The results show that 60% of strains weremecA positive when tested by PCR although 25.7% of strains were oxacillin resistant when tested with ATB STAPH. Qualitative slime production ofS. aureus using CRA revealed that 74.3% ofS. aureus were slime producers. All the strains carried theica gene.     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

Disappearance of differences in antibiotic resistance of Staphylococcus aureus strains isolated in hospitals and in general practice

There is general opinion that Staphylococcus aureus strains isolated in hospitals are more frequently resistant to antibiotics than community strains, however, the increasing resemblance between hospital and community strains has been recently reported. The aim of the study was to compare the antibiotic resistance and phage-type pattern of S. aureus strains isolated from patients treated either in hospitals or in general practice in northern part of Poland. The study was conducted on 771 S. aureus strains isolated from different specimens. Phage typing was performed according to the method of Blair and Williams. The drug susceptibility was determined by the disc-diffusion method. There were no significant differences in antibiotic resistance or phage-type pattern when hospital and community methicillin-sensitive S. aureus (MSSA) strains were compared. The most MSSA were resistant to penicillin (84.6% and 82.1% respectively) and doxycycline (49.3% and 50.4% respectively) whereas they were rarely resistant to other antibiotics. The predominance of phage group II was found in both hospitals (28.0%) and general practice (29.9%). Phage group III, usually associated with hospitals, occurred in small percentage (12.9% and 9.4% respectively) while to this group predominantly (76.6%) multiresistant methicillin resistant S. aureus (MRSA) isolated in hospitals belonged. These results suggest, that there is only slight difference in antibiotic resistance between hospital and community S. aureus strains. Antibiotic resistance pattern mainly results from frequency of appearance of MRSA, mostly occurring in hospitals.     

Quantitative disk diffusion as a convenient method for determining minimum inhibitory concentrations of oxacillin for staphylococci strains

In this study the susceptibility of 58 coagulase-negative staphylococci (CoNS) strains and 58 Staphylococcus aureus strains to oxacillin was evaluated by a novel method called quantitative disk diffusion (DD) method. The results obtained were compared to phenotypic methods as agar dilution (AD) for oxacillin, disk diffusion (DD) for cefoxitin, and related to the presence of the mecA gene detected by PCR. Minimum inhibitory concentrations (MIC) determined by the quantitative DD method were equivalent to MICs determined in the AD method for S. aureus (Student's t test, p=0.99) and CoNS (Student's t test, p=0.97). Incongruent results between PCR mecA gene determinations and the quantitative DD method were obtained in 8 strains (5 S. aureus and 3 CoNS) where the mecA gene expression was blocked. However, oxacillin resistance was detected by the proposed method even in staphylococci strains showing low-level or heterogeneous resistance to the antibiotic while other phenotypic methods failed. The single quantitative DD method is not expensive, it can be performed in any laboratory and permits accurate identification of oxacillin resistant staphylococci.     

Identification and discrimination of Staphylococcus aureus strains using matrix-assisted laser desorption/ionization-time of flight mass spectrometry

Staphylococcus aureus is an important human pathogen frequently resistant to a wide range of antibiotics. Methicillin-resistant S. aureus (MRSA) strains are common nosocomial pathogens that pose a world-wide problem. Rapid and accurate discrimination between methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus is essential for appropriate therapeutic management and timely intervention for infection control. We report here the application of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for monitoring the bacterial fingerprints expressed by two well characterized S. aureus strains ATCC 29213 (MSSA) and ATCC 43330 (MRSA). Consistent strain-specific data were obtained from subcultures analyzed over a period of three months as well as after changing the growth media from Mueller-Hinton to blood agar indicating the reliability of the method. The bacterial fingerprints of these two strains were compared to independent clinical isolates of S. aureus. A uniform signature profile for MRSA could not be identified. However, the bacterial fingerprints obtained proved to be specific for any given strain. This study demonstrates that MALDI-TOF MS is a powerful method for rapid identification of clonal strains of S. aureus, which might be useful for tracking nosocomial outbreaks of MRSA and for epidemiologic studies of infections diseases in general.     

Sensitivity of Staphylococcus aureus strains isolated from the patients of the SP ZOZ Hospital in Nidzica to bacteriocidal agents

The aim of this study was to evaluate a frequency of isolation and antimicrobial susceptibility testing of Staphylococcus aureus strains isolated from clinical specimens obtained from patients hospitalized in different hospital wards (SP ZOZ) in Nidzica from 01. 09. 2000 to 31. 12. 2003. During over three years 716 Staphylococcus aureus strains were cultured out of 15517 clinical specimens supplied to the Bacteriological Laboratory of SP ZOZ in Nidzica. S. aureus strains were isolated from 4.6% of examined samples. Samples were collected from patients hospitalized in all wards (five wards). Analysis of susceptibility to antimicrobial agents of identified S. aureus strains was performed. Seventy strains (9.8%) were metihicillin-resistant (MRSA). One hundred twenty four strains (17.3%) revealed inducible resistance to macrolides, linkosamides and streptogramins B (MLS, mechanism). The greatest activity in vitro against clinical S. aureus strains showed glycopeptide antibiotic--vancomycin (100% of susceptible strains). Clinical S. aureus strains isolated from patients of hospital in Nidzica are in the majority susceptible to antibiotics/chemotherapeutics, except of penicillin. Percentage of methicillin-resistant strains (MRSA) is not high (<10). Nevertheless, constant monitoring of a drug susceptibility of nosocomial S. aureus strains is important, considering the necessity of control of current epidemiological and therapeutic situation.     

Evaluation of susceptibility to antibiotics of Staphylococcus aureus strains resistant to methicillin]

Methicillin-resistant strains of Staphylococcus aureus (MRSA) constitute a serious diagnostic and therapeutic problem. Over 500 strains of Staphylococcus aureus were tested for susceptibility to methicillin. By application of a screening method, 13.7% of these strains were classified as methicillin-resistant. Over 95% of these strains were isolated from hospital infections. Applying criteria of belonging of these strains to methicillin-resistance classes it was found that 49.3% belonged to class II, 31.2% to class III and 19.5% to class IV. Analysis of susceptibility to antibiotics of MRSA strains demonstrated significant differences between class II and between class III and IV in resistance to imipenem, gentamycin, erythromycin and tetracycline. All tested strains were susceptible to ciprofloxacin, ofloxacin, vancomycin and teicoplanin. The screening method (25 mg methicillin/l of TSA medium) results in obtaining of univocal results of determination of methicillin-resistance in S. aureus.     

Direct Quantitation of the Numbers of Individual Penicillin-Binding Proteins per Cell in Staphylococcus aureus

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in bacterial peptidoglycan assembly. The absolute numbers of each PBP were determined by direct measurement and have been reported for two Staphylococcus aureus strains, RN4220 (methicillin-sensitive S. aureus) and RN450M (methicillin-resistant S. aureus). From the specific activity of the labeled penicillin and the absolute number of disintegrations per minute, and from the number of CFU per milliliter calculated from proteins and optical density, a determination of the number of PBPs per cell was made. These numbers ranged from approximately 150 to 825 PBPs/cell and represent the first direct determination of absolute numbers of PBPs in S. aureus.     

Molecular genetic typing of methicillin-resistant Staphylococcus aureus strains, isolated in hospitals of different regions of the Russia and Belarus

The study of the length of the amplification products of the coagulase gene with the subsequent restriction analysis (the method of PCR--restrictive fragment length polymorphism, or RFLP) was used for typing 90 S. aureus strains. Among the strains under study, 78 were methicillin-resistant S. aureus (MRSA) strains, including 74 obtained in 1986 - 2002 in hospitals of different cities of the Russia and Belarus, as well as epidemic strains EMRSA-1, -2, -3, -12, obtained from the National Laboratory of Health, London (UK). The use of this method made it possible to type all the strains under study, which were differentiated into 9 groups by means of endonuclease Sfo1 and 7 groups by means of Alu1. Majority of clinical MRSA strains, belonged, according to the type of restriction, to groups 4 and 5. The study of the coagulase gene by the method of PCR - RFLP made it possible: to analyze the epidemic situation in hospitals for a period of several years; to compare the properties of strains isolated in different hospitals; to establish the genetic relationship of strains, isolated in 1998 - 2002, with strains, isolated in 1986 - 1990. The results of the study suggest that at least two epidemic MRSA strains, genetically similar to international strains, circulate in hospitals of Russia.     

Antibiotic resistance of staphylococci isolated from outpatients

The aim of the study was to determine susceptibility of 587 strains of S. aureus and 85 strains of coagulase-negative staphylococci isolated from outpatients in Poznań to co-trimoxazole, amoxycillin/clavulanic acid, erythromycin, gentamycin, doxycycline, ampicillin, oxacillin, cephradine, clindamycin and neomycin. Also methicillin-resistant strains were determined as well as strains ability to produce beta-lactamases. Susceptibility testing and examination of methicillin-resistant strains were performed by the disc diffusion techniques according to recommendation of NCCLS. Methicillin-resistant strains were additionally examined to their sensitivity to vankomycin and teicoplanin. beta-lactamase production was detected using nitrocefin impregnated discs and iodometric method. Amoxacillin/clavulanic acid, gentamycin, co-trimoxazole, cephradin, oxacillin and clindamycin occurred to be very active against both, S. aureus and coagulase-negative staphylococci. 84.7% to 100% of examined strains were sensitive to these drugs. Doxycyclin, erythromycin and ampicillin were less effective. Nine strains (1.5%) of 587 strains of S. aureus as well as 7 strains (8.7%) of coagulase-negative staphylococci were methicillin-resistant. All of methicillin-resistant strains were sensitive to vancomycin and teicoplanin. More than 75% of S. aureus and close to 50% of coagulase-negative staphylococci were able to produce beta-lactamases.     

Methicillin-resistant Staphylococcus aureus (MRSA) with high resistance to mupirocin in hospitals of the Gdańsk region

Occurrence of high-level mupirocin resistance in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from 18 hospitals in Gdańsk area was determined. The study was carried out on 190 MRSA isolated in 1997-2000 from various clinical samples. The strains were tested for high-level mupirocin resistance by 200 micrograms mupirocin disc. The minimum inhibitory concentrations (MIC) for methicillin were estimated by agar dilution. Sensitivity to other antibiotics was determined in disc-diffusion method and to vancomycin in agar dilution method additionally. The strains were typed by set of 10 experimental phages and compared by the method of PCR-RFLP analysis of coagulase gen restriction fragment length polymorphism. There were low frequency of high-level mupirocin resistance in MRSA strains (4.7%) that were found only in 3 hospitals, in 6 patients. All of them were high-resistant also to methicillin and resistant to doxycyclin, gentamycin, erytromycin, klindamycin, ciprofloksacin, rifampicin, resistant or intermediate sensitive to fusidic acid but sensitive to vancomycin, teikoplanin and bacitracin. The origin all of the MRSA strains high-resistant to mupirocin probably was the same, except one strain, because they were belonged to one genetic type and possessed the same phage pattern.     

Antibiotic susceptibility and occurrence of ESBL in Pseudomonas aeruginosa strains isolated from different clinical specimens

The aim of this study was to evaluate the drug susceptibility of 132 P. aeruginosa strains isolated from patients hospitalized in SPSK University Hospital in Bialystok. The isolates were obtained from clinical specimens over an 11-month period in 2001 and 2002. All the strains were identified in automatic ATB system using API 20 NE strips, and their susceptibility to antibiotics was tested by standard disc-diffusion method and agar dilution method. The minimal inhibitory concentration (MIC) was determined for five antibiotics: piperacillin, amikacin, ceftazidime, imipenem and ciprofloxacin. The majority of strains were susceptible to ceftazidime (91.7%), piperacillin combined with tazobactam (85.6%), amikacin (80.3%), meropenem and imipenem (81.8%). Many of our strains were resistant to cefotaxime (73.5%), ticarcillin (53%) and ciprofloxacin (48.5%). Also, the trial was undertaken to detect strains producing extended-spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) among P. aeruginosa rods isolated from different specimens. ESBL-producing strains were detected with double disc test (DDST) and combination double disc (CD) test. Clavulanate was applied as the inhibitor of these beta-lactamases. Strains producing ESBL were not found. On the other hand, as many as 127 P. aeruginosa strains (96.2%) produced inducible beta-lactamases (IBL).     

Phage types of Staphylococcus aureus strains and their antibiotic resistance in carriers of medical students population

The phage types of 78 S. aureus strains isolated from nose swabs obtained from a medical students in 2005 -2006 was determined and antibiotic resistance of the phage types was analysed. 680 students were tested in order to obtain the strains and 11.5% of them were carriers of S. aureus. Phage typing was performed using basic set of23 phages and 3 additional phages: 88, 89 and 187. Drug resistance was determined by the disc-diffusion method. The most frequent in studied population were the group III (21.8%) and strains lysed by phages belonging to varied groups (21.8%). Highly different phage patterns were observed among strains belonging to each of the group. Strains belonging to the group III as the strains lysed by phages from varied groups were most frequently resistant only to penicillin (52,9% respectively). Resistance to penicillin was also most often observed in the strains belonging to another groups and phage types. Usefulness of the additional phages 88,89 and 187 was in the investigations as no more than 51% of strains was lysed by this phages.     

Susceptibility to selected chemotherapeutics of Staphylococcus aureus strains resistant to methycyline isolated from clinical materials in the years 1991-1992 and 1997

The susceptibility to selected chemotherapeutic agents was determined in 100 strains of Staphylococcus aureus methicillin-resistant (MRSA) isolated from clinical materials in 1991-1992 (50 strains) and in 1997 (50 strains). Two methods were used for the determination: disc method and antibiotic dilution in agar. The minimal inhibitory concentration (MIC) was determined for vancomycin, teicoplanin, furazolidone, nitrofurantoin, ofloxacin, gentamicin, netilmicin and trimethoprim. The concentrations of the chemotherapeutics in the substrate ranged from 0.125 to 512 mg/l. The obtained results served for drawing of the following conclusions: all studied MRSA strains isolated in 1991-1992 and in 1997 were sensitive to glycopeptide antibiotics: vancomycin and teicoplanin, to nitrofurans: nitrofurantoin and furazolidone, and to fusidic acid. MRSA strains isolated in 1991-1992 were sensitive to ofloxacin, but in 1997 about 80% of the strains were resistant to that antibiotic, and this resistance was noted in S. aureus strains with homogeneous resistance to methicillin. Increasing frequency of resistance to mupirocin was found, in 1991-1992 4% of the strains were resistant, and in 1997 the resistance of MRSA to that antibiotic was found in 12%. No changes occurred in the sensitivity of staphylococci to trimethoprim/sulfamethoxazole (cotrimoxazole). About 94% of strains in 1991-1992 and 1997 were sensitive to that drug. The sensitivity to cotrimoxazole is connected with one of its components (trimethoprim), with 94% of MRSA strains sensitive to it.     

Use of new methods for detection of beta-lactamases with extended-spectrum substrates (ESBLs)

The aim of performed examinations was to compare results of two methods applied for the detection of extended-spectrum beta-lactamases (ESBLs). Two hundred and sixty strains of Gram-negative rods were cultured from clinical specimens obtained from hospitalized patients. These strains were identified as ESBL-positive on the basis of the double-disc method (DDST) according to Jarlier et al. (1988) results. Identification of strains was performed in automatic ATB Expression system (bioMerieux, France). All strains were determined using a novel test for detection of ESBL-type enzymes (DD) according to Appleton (1999). Two discs are applied in this test: with cefpodoxime (CPD) and with cefpodoxime/clavulanate (CD 01, diagnostic disc). Consistent results of two methods (DDST and DD) were obtained in the case of 166 from among 260 of examined strains (60.4%). Consistent results concerned 161 out of 222 examined strains of enteric rods (72.5%) and only 5 from among 38 of other strains (mostly belonging to the group of non-fermenting rods). On the basis of performed investigations it can be stated that the novel method of extended-spectrum beta-lactamases (ESBLs) detection (DD) according to Appleton (1999) is more objective and easier for interpretation than the double-disc synergy test (DDST) according to Jarlier et al. (DDST), which is widely applied in the routine microbiological diagnostics.