A Rapid Latex Agglutination Assay for the Detection of Penicillin-Binding Protein 2′

A simple and rapid slide latex agglutination assay was developed to detect penicillin-binding protein 2′ (PBP2′) from isolates of staphylococi. PBP2′ present in the membranes of methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant coagulase negative staphylococci (MRCNS) was rapidly extracted by alkaline treatment and, by combining with a slide agglutination reaction using latex particles sensitized with monoclonal antibodies raised against it, PBP2′ could be detected from a single loopful of cells taken from agar plates not containing beta-lactum antibiotics within 15 min. In a study of clinical isolates previously characterized as either MRSA or methicillin-susceptible Staphylococcus aureus (MSSA) by antibiotic susceptibility testing, 231 specimens of 232 MRSA were PBP2′ positive by latex agglutination, and the 87 specimens of MSSA were all negative. One specimen identified as MRSA by susceptibility testing but PBP2′ negative by latex agglutination was confirmed as mecA gene negative by PCR. This simple and rapid slide latex reagent should be useful in clinical diagnostics.     

Properties of a Novel PBP2A Protein Homolog from Staphylococcus aureus Strain LGA251 and Its Contribution to the ��-Lactam-resistant Phenotype

Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-to-strain variation in resistance level, in genetic background, and also in the structure of the chromosomal cassette (SCCmec) that carries the resistance gene mecA. In contrast, strain-to-strain variation in the sequence of the mecA determinant was found to be much more limited among MRSA isolates examined so far. The first exception to this came with the recent identification of MRSA strain LGA251, which carries a new homolog of this gene together with regulatory elements mecI/mecR that also have novel, highly divergent structures. After cloning and purification in Escherichia coli, PBP2A_LGA, the protein product of the new mecA homolog, showed aberrant mobility in SDS-PAGE, structural instability and loss of activity at 37 °C, and a higher relative affinity for oxacillin as compared with cefoxitin. The mecA homolog free of its regulatory elements was cloned into a plasmid and introduced into the background of the β-lactam-susceptible S. aureus strain COL-S. In this background, the mecA homolog expressed a high-level resistance to cefoxitin (MIC = 400 μg/ml) and a somewhat lower resistance to oxacillin (minimal inhibitory concentration = 200 μg/ml). Similar to PBP2A, the protein homolog PBP2A_LGA was able to replace the essential function of the S. aureus PBP2 for growth. In contrast to PBP2A, PBP2A_LGA did not depend on the transglycosylase activity of the native PBP2 for expression of high level resistance to oxacillin, suggesting that the PBP2A homolog may preferentially cooperate with a monofunctional transglycosylase as the alternative source of transglycosylase activity.     

Healthcare-associated (HA) and community-associated (CA) methicillin resistant Staphylococcus aureus (MRSA) in Bangladesh – Source,diagnosis and treatment

Methicillin-resistant Staphylococcus aureus (MRSA) has long been a common pathogen in healthcare facilities, but now, it has emerged as a problematic pathogen in the community setting as well. This study reported source, diagnosis and treatment of HA-MRSA and CA-MRSA.A total of sixty-five clinical samples (urine, pus, wound swab) were collected from clinical origin of Dhaka city, Bangladesh. All the isolates were tested phenotypically by conventional methods and genotypically by PCR targeting nuc, pvl and mecA genes. Finally sequencing was carried out for pvl gene to know the mutagenic variation or any amino acid changes in pvl gene. Chi square test was employed for statistical analysis. Patients of age group 51–60?years are more susceptible (46.15%) to MRSA, CA-MRSA or HA-MRSA infection. Female are (32.30%) more susceptible to MRSA infection. Among 65 isolates 53 isolates identified phenotypically as S. aureus. These were positive for amplification of nuc (270?bp) gene of S. aureus. Moreover, among 53 isolates 33 phenotypically considered as MRSA and 38 (72%) showed positive amplification for mecA (162?bp) gene. Among 38 MRSA isolates 22 (57.89%) confirmed as CA-MRSA and 16 (42.10%) as HA-MRSA. Finally, sequence analysis for lukS/F-PV genes from 4 representative isolates detected a new single nucleotide polymorphism in comparison with the control sequence. However, no amino acid changes were found. Statistical analysis showed HA-MRSA isolates were more commonly found in urine sample and CA-MRSA in pus and wound swab. CA-MRSA isolates were more resistant to tested antibiotics than HA-MRSA.     

Comparison of <Emphasis Type="Italic">mecA</Emphasis> Polymerase Chain Reaction With Phenotypic Methods for the Detection of Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In the present study, several conventional methods to detect methicillin-resistant Staphylococcus aureus (MRSA) were compared with polymerase chain reaction (PCR) detection of mecA gene–positive isolates. Cefoxitin E-test was also evaluated as a possible phenotypic method of MRSA detection. Oxacillin agar screen and PBP2′ latex agglutination methods were found to be more sensitive than oxacillin and cefoxitin disk-diffusion methods. Cefoxitin disk diffusion was found to be the most specific. A combination of oxacillin agar screening with cefoxitin disk diffusion, or oxacillin disk diffusion with PBP2′, improved sensitivity and specificity. Cefoxitin E-test with the current break points had low sensitivity and specificity (33.3% and 75%, respectively) for the detection of MRSA. However, changing the break points to ≤ 4 μg/ml and to ≥ 6 μg/ml for sensitive and resistant, respectively, greatly improved both. Changing the 30-μg cefoxitin disk-diffusion break points to ≤ 21 mm for resistant slightly improved sensitivity but had no effect on specificity. It was therefore concluded that the use of more than one screening method is necessary to detect all MRSA isolates in clinical settings.     

Phage typing,PCR amplification for mecA gene,and antibiotic resistance patterns as epidemiologic markers in nosocomial outbreaks of methicillin resistant Staphylococcus aureus

Staphylococcus aureus is one of the major causes of community and hospital-acquired infections. Bacteriophage considered as a major risk factor acquires S. aureus new virulence genetic elements. A total number of 119 S. aureus isolated from different specimens obtained from (RKH) were distinguished by susceptibility to 19 antimicrobial agents, phage typing, and PCR amplification for mecA gene. All of MRSA isolates harbored mecA gene, except three unique isolates. The predominant phage group is belonging to the (mixed group). Phage group (II) considered as an epidemiological marker correlated to β-lactamase hyper producer isolates. MRSA isolates indicated high prevalence of phage group (II) with highly increase for phage types (Ø3A), which were correlated to the skin. Phage types (Ø80/Ø81) played an important roll in Community Acquired Methicillin Resistant S. aureus (CAMRSA). Three outpatients MRSA isolates had low multiresistance against Bacitracin (Ba) and Fusidic acid (FD), considered as CAMRSA isolates. It was detected that group I typed all FD-resistant MSSA isolates. Phage groups (M) and (II) were found almost to be integrated for Gentamycin (GN) resistance especially phage type (Ø95) which relatively increased up to 20% in MRSA. Tetracycline (TE) resistant isolates typed by groups (II) and (III) in MSSA. Only one isolate resistant to Sulphamethoxazole/Trimethoprim (SXT) was typed by (III/V) alone in MSSA. MRSA isolates resistant to Chloramphenicol (C) and Ba were typed by all groups except (V). It could be concluded that (PERSA) S. aureus isolates from the wound that originated and colonized, and started to build up multi-resistance against the topical treatment antibiotics. In this study, some unique sporadic isolates for both MRSA and MSSA could be used as biological, molecular and epidemiological markers such as prospective tools.     

Comparison of Real-Time PCR with Disk Diffusion, Agar Screen and E-test Methods for Detection of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the “gold standard” comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.     

Exposure of Clinical MRSA Heterogeneous Strains to β-Lactams Redirects Metabolism to Optimize Energy Production through the TCA Cycle

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important pathogens both in health care and community-onset infections. The prerequisite for methicillin resistance is mecA, which encodes a β-lactam-insensitive penicillin binding protein PBP2a. A characteristic of MRSA strains from hospital and community associated infections is their heterogeneous expression of resistance to β-lactam (HeR) in which only a small portion (≤0.1%) of the population expresses resistance to oxacillin (OXA) ≥10 µg/ml, while in other isolates, most of the population expresses resistance to a high level (homotypic resistance, HoR). The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a β-lactam-mediated SOS response and related lexA and recA genes. In the present study we investigated the cellular physiology of HeR-MRSA strains during the process of β-lactam-mediated HeR/HoR selection at sub-inhibitory concentrations by using a combinatorial approach of microarray analyses and global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) to investigate changes in metabolic pathways and the metabolome associated with β-lactam-mediated HeR/HoR selection in clinically relevant heterogeneous MRSA. We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways. Inactivation of the TCA cycle enzyme cis-aconitase gene in the SA13011-HeR strain abolished β-lactam-mediated HeR/HoR selection demonstrating the significance of altered TCA cycle activity during the HeR/HoR selection. These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to β-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of β-lactam antibiotics.     

Methicillin (Oxacillin)-Resistant Staphylococcus aureus Strains Isolated from Major Food Animals and Their Potential Transmission to Humans

From May 2001 to April 2003, various types of specimens from cattle, pigs, and chickens were collected and examined for the presence of methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA). S. aureus was isolated and positively identified by using Gram staining, colony morphology, tests for coagulase and urease activities, and an API Staph Ident system. Among 1,913 specimens collected from the animals, 421 contained S. aureus; of these, 28 contained S. aureus resistant to concentrations of oxacillin higher than 2 μg/ml. Isolates from 15 of the 28 specimens were positive by PCR for the mecA gene. Of the 15 mecA-positive MRSA isolates, 12 were from dairy cows and 3 were from chickens. Antimicrobial susceptibility tests of mecA-positive MRSA strains were performed by the disk diffusion method. All isolates were resistant to members of the penicillin family, such as ampicillin, oxacillin, and penicillin. All isolates were also susceptible to amikacin, vancomycin, and trimethoprim-sulfamethoxazole. To determine molecular epidemiological relatedness of these 15 animal MRSA isolates to isolates from humans, random amplified polymorphic DNA (RAPD) patterns were generated by arbitrarily primed PCR. The RAPD patterns of six of the isolates from animals were identical to the patterns of certain isolates from humans. The antibiotypes of the six animal isolates revealed types similar to those of the human isolates. These data suggested that the genomes of the six animal MRSA isolates were very closely related to those of some human MRSA isolates and were a possible source of human infections caused by consuming contaminated food products made from these animals.     

Isolation and characterization of methicillin-resistantStaphylococcus aureus from livestock and poultry meat

Meat samples from sheep, bovine, camel and poultry were collected from Amman area and were processed and tested for the presence of methicillin (oxacillin) resistantStaphylococcus aureus (MRSA). Identity ofS. aureus was ensured by Gram-staining and a battery of biochemical tests. From 1260 meat samples, 157S. aureus positive isolates were identified. Of the 157 isolates, 30 were resistant to methicillin levels greater than 2 μg/ml and only 15 weremecA-positive MRSA originating mainly from sheep and chicken. Subjecting themecA-positive MRSA to antibiotic susceptibility testing revealed that all isolates were resistant to β-lactam antibiotics (ampicillin, penicillin, and oxacillin) and were sensitive to vancomycin, trimethoprim, chloramphenicol and cephalothin. Randomamplified polymorphic DNA (RAPD) analysis ofmecA-positive animal isolates generated six different patterns. Comparing these results with results of isolates of human origin of our laboratory there is some molecular epidemiological relatedness between both and could be a possible source of infections through consuming contaminated meat products, direct contact or meat processing.     

The Mechanism of Heterogeneous Beta-Lactam Resistance in MRSA: Key Role of the Stringent Stress Response

All methicillin resistant S. aureus (MRSA) strains carry an acquired genetic determinant – mecA or mecC - which encode for a low affinity penicillin binding protein –PBP2A or PBP2A′ – that can continue the catalysis of peptidoglycan transpeptidation in the presence of high concentrations of beta-lactam antibiotics which would inhibit the native PBPs normally involved with the synthesis of staphylococcal cell wall peptidoglycan. In contrast to this common genetic and biochemical mechanism carried by all MRSA strains, the level of beta-lactam antibiotic resistance shows a very wide strain to strain variation, the mechanism of which has remained poorly understood. The overwhelming majority of MRSA strains produce a unique – heterogeneous – phenotype in which the great majority of the bacteria exhibit very poor resistance often close to the MIC value of susceptible S. aureus strains. However, cultures of such heterogeneously resistant MRSA strains also contain subpopulations of bacteria with extremely high beta-lactam MIC values and the resistance level and frequency of the highly resistant cells in such strain is a characteristic of the particular MRSA clone. In the study described in this communication, we used a variety of experimental models to understand the mechanism of heterogeneous beta-lactam resistance. Methicillin-susceptible S. aureus (MSSA) that received the mecA determinant in the laboratory either on a plasmid or in the form of a chromosomal SCCmec cassette, generated heterogeneously resistant cultures and the highly resistant subpopulations that emerged in these models had increased levels of PBP2A and were composed of bacteria in which the stringent stress response was induced. Each of the major heterogeneously resistant clones of MRSA clinical isolates could be converted to express high level and homogeneous resistance if the growth medium contained an inducer of the stringent stress response.     

The prevalence of Staphylococcus aureus and methicillin resistant Staphylococcus aureus in milk and dairy products in Riyadh,Saudi Arabia

In terms of life- menaced contagion, methicillin resistant Staphylococcus aureus (MRSA) is known to be one of which and it is truly notable in the contaminated food causing a community health anxiety. However, the occurrence of S. aureus and MRSA in diverse kinds of dairy products have been tested in this study. Samples from: raw milk (unpasteurized) from horse, goat, camel, and cow origins and unpacked cheese were checked for the recovered strains of such bacterium and MRSA. Wholly, MRSA isolates were verified for antimicrobial susceptibility and further characterized by mecA and staphylococcal cassette chromosome mec (SCCmec) typing. Also, Panton-Valentine Leukocidin (PVL), Staphylococcus aureus protein A (spa), and Staphylococcal enterotoxins (SEs) were also tested between all positive MRSA isolates in order to discover the virulence factors. Consequently, 70% of the 100 collected dairy products samples were contaminated by S. aureus bacteria and 72.9% of them were defined as MRSA. 9.8% of MRSA isolates contained mecA genes with SCCmec type II (80%) as the most common SCCmec type. Moreover, large number of MRSA isolates were identified as multidrug resistance and 28.6% of MRSA-mecA positive isolates were also carried vancomycin resistance genes (i.e., vanB). Too, spa gene was detected between 9.8% of MRSA isolates but PVL gene was not spotted at all. Additionally, the existing of SEs was variable between MRSA isolates and the most common type was SEH (51%). In general, our results confirmed that raw milk and unpacked cheese in the Kingdom of Saudi Arabia (Riyadh) is a potential vehicle for multidrug resistant MRSA transmission. It is a critical civic health menace and stresses, thus; the need of applying well cleanliness practices is essential.     

Penicillin-binding proteins and cell wall composition in beta-lactam-sensitive and -resistant strains of Staphylococcus sciuri

A close homologue of the acquired Staphylococcus aureus mecA gene is present as a native gene in Staphylococcus sciuri. We determined the patterns of penicillin-binding proteins (PBPs) and the peptidoglycan compositions of several S. sciuri strains to explore the functions of this mecA homologue, named pbpD, in its native S. sciuri environment. The protein product of pbpD was identified as PBP4 with a molecular mass of 84 kDa, one of the six PBPs present in representatives of each of three subspecies of S. sciuri examined. PBP4 had a low affinity for nafcillin, reacted with a monoclonal antibody raised against S. aureus PBP2A, and was greatly overproduced in oxacillin-resistant clinical isolate S. sciuri SS37 and to a lesser extent in resistant laboratory mutant K1M200. An additional PBP inducible by oxacillin and corresponding to S. aureus PBP2A was identified in another oxacillin-resistant clinical isolate, S. sciuri K3, which harbors an S. aureus copy of mecA. Oxacillin resistance depended on the overtranscribed S. sciuri pbpD gene in strains SS37 and K1M200, while the resistance of strain K3 depended on the S. aureus copy of mecA. Our data provide evidence that both S. aureus mecA and S. sciuri pbpD can function as resistance determinants in either an S. aureus or an S. sciuri background and that the protein products of these genes, S. aureus PBP2A and S. sciuri PBP4, can participate in the biosynthesis of peptidoglycan, the muropeptide composition of which depends on the bacterium “hosting” the resistance gene.     

Characterization of Methicillin-Resistant and -Susceptible Staphylococcal Isolates from Bovine Milk in Northwestern China

Emergence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) in bovine milk is a major public health concern. The primary purpose of this research was to determine molecular genetic characteristics and antibiotic resistance of staphylococcal isolates recovered from milk of mastitic cows in the Shaanxi Province in Northwestern China. One hundred and thirteen methicillin-susceptible Staphylococcus aureus (MSSA), one mecA-positive and phenotype-positive MRSA, seven mecA- and mecC- negative but phenotype-positive MRSA and two MR-CoNS including one oxacillin-susceptible mecA-positive Staphylococcus haemolyticus (OS-MRSH) and one mecA-positive and methicillin-resistant Staphylococcus epidermidis (MRSE) isolates were recovered from 214 quarter milk samples on 4 dairy farms. All above 123 isolates were subjected to antibiotic resistance profiling. S. aureus isolates were also genotyped using the spa typing and the multilocus sequence typing (MLST). Eight MRSA and 2 MR-CoNS isolates were additionally tested for SCCmec types. Resistance was common among isolates against ampicillin or penicillin (80.5%), kanamycin (68.3%), gentamicin (67.5%), tetracycline (43.9%) and chloramphenicol (30.1%). However, no isolate was resistant to vancomycin or teicoplanin. Twenty, 29 and 58 isolates showed resistance to 1, 2 or more than 2 antibiotics, respectively. The predominant multidrug resistance profile was penicillin/ampicillin/kanamycin/gentamicin/tetracycline (46 isolates). Most S. aureus isolates belonged to spa types t524 (n = 63), t11772 (a new type, n = 31) and t4207 (n = 15). At the same time, MLST types ST71 (n = 67) and ST2738 (a new type, n = 45) were identified as dominant sequence types. The mecA-positive and phenotype-positive MRSA isolate had a composite genotype t524-ST71-SCCmecIVa, while 7 mecA-negative but phenotype-positive MRSA isolates were all t524-ST71. The OS-MRSH isolate contained a type V SCCmec cassette, while the MRSE isolate possessed a non-typeable SCCmec. The spa-MLST types t11772-ST2738 (n = 27), t11807-ST2683 (n = 4) and t11771-ST2738 (n = 3) were newly identified genotypes of S. aureus. These new genotypes and multidrug-resistant staphylococci could pose additional threat to animal and human health.     

Characterization of Staphylococcus aureus from Humans and a Comparison with ?solates of Animal Origin,in North Dakota,United States

Different clones of methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus have been found in humans as well as in animals and retail meat. However, more information about the genetic characteristics and similarities between strains is needed. The aim of this study was to identify and characterize Staphylococcus aureus from humans, and to compare their characteristics with isolates of animal origin. A total of 550 nasal swabs were taken from healthy humans, and S. aureus was isolated and identified. Positive S. aureus isolates were subjected to molecular typing and susceptibility testing. In addition, 108 MRSA isolates recovered from clinical patients in the state of North Dakota and 133 S. aureus isolates from animals and meat previously analyzed were included. The nasal carriage of S. aureus in healthy people was 7.6% and, in general, clones were genetically diverse. None of the S. aureus strains obtained from healthy people were mecA- or PVL-positive. A total of 105 (97.2%) MRSA isolates from clinical cases harbored the mecA gene and 11 (10.2%) isolated from blood stream infections harbored the PVL gene. The most common resistance profile among S. aureus from healthy people was penicillin, and from clinical cases were erythromycin-penicillin-ciprofloxacin. The rate of multidrug resistance (MDR) was 70% in humans. Most of the S. aureus harboring mecA and PVL genes were identified as ST5 and ST8, and exhibited MDR. However, S. aureus isolates of animal origin used for comparison exhibited a lower rate of MDR. The most common resistance profiles in isolates of animal origin were penicillin-tetracycline and penicillin-tetracycline-erythromycin, in animals and raw meat, respectively. The ST5 was also found in animals and meat, with ST9 and ST398 being the major clones. The genetic similarity between clones from humans and meat suggests the risk of spread of S. aureus in the food chain.     

Variability in SCC <Emphasis Type="Italic">mec</Emphasis><Subscript>N1</Subscript> spreading among injection drug users in Zurich,Switzerland

Background  

An extremely low level methicillin resistant Staphylococcus aureus (MRSA) belonging to ST45, circulates among intravenous drug users in the Zurich area. This clone can be misinterpreted as an MSSA by phenotypic oxacillin resistance tests, although it carries a staphylococcal cassette chromosome mec (SCCmec) element encoding a functional mecA gene and it produces PBP2a.     

Staphylococcus aureus Keratitis: A Review of Hospital Cases

Background

Methicillin-resistant Staphylococcus aureus (MRSA) infection is an important public health issue. The study aimed to characterize the patient demographics, clinical features, antibiotic susceptibility, and clinical outcomes of keratitis caused by S. aureus, and to make a comparison between MRSA and methicillin-sensitive S. aureus (MSSA) isolates.

Methodology/Principal findings

Patients (n = 59) with culture-proven S. aureus keratitis treated in Chang Gung Memorial Hospital between January 1, 2006, and December 31, 2010, were included in our study. Patients'' demographic and clinical data were retrospectively reviewed. Twenty-six MRSA (44%) and 33 MSSA (56%) isolates were collected. The MRSA keratitis was significantly more common among the patients with healthcare exposure (P = 0.038), but 46.2% (12/26) of patients with MRSA keratitis were considered to have community-associated infections. All isolates were susceptible to vancomycin. MRSA isolates were significantly more resistant to clindamycin, erythromycin, and sulfamethoxazole/trimethoprim. Ocular surface disease was a significant risk factor for MRSA keratitis (P = 0.011). Visual outcome did not differ significantly between the MRSA and MSSA groups. However, age (B = 0.01, P = 0.035, 95% confidence interval [CI]: 0.001–0.019) and visual acuity at presentation (B = 0.749, P<0.001, 95% CI: 0.573–0.926) were significantly correlated with visual outcome.

Conclusions/Significance

Ocular surface disease is an important predisposing factor for S. aureus keratitis, especially for MRSA infections. Advanced age and poor visual acuity at presentation are important prognostic indicators for poor visual outcome in S. aureus keratitis. Oxacillin resistance may not be a significant prognostic indicator.