A rapid method for the detection of methicillin resistance in staphylococci was developed. The method was based on the polymerase chain reaction (PCR) and primers that targeted the internal region of the coding frame of the mec gene. The amplification reaction was carried out with crude cell lysates as a source of target DNA and provided data in less than 5 h. Seventy-four isolates of coagulase-negative staphylococci were tested by PCR, DNA hybridization with a probe derived from the mec gene, and an agar dilution susceptibility assay.PCR results showed a 100% correlation with the susceptibility assay carried out with high inocula (108 CFU) and incubation at 32°C for 48 h. PCR was more sensitive and specific than DNA hybridization in detecting methicillin resistance in coagulase-negative staphylococci. The former technique identified the mec gene in all the strains which were phenotypically resistant but which did not hybridize with the probe. Identification of methicillin-resistant strains by PCR offers a very specific, sensitive, and rapid alternative to traditional susceptibility tests and DNA hybridization as a guide for the treatment of infections caused by staphylococci.Several groups of investigators have described DNA probes that can be successfully used to detect methicillinresistant staphylococci (1,5,12).These probes are specific fragments generated by restriction endonuclease digestion (1, 5) or are polymerase chain reaction (PCR)-generated fragments (12) of the gene which, in both coagulase-positive and -negative staphylococci, encodes for the synthesis of a new penicillin-binding protein (PBP 2a) which mediates clinically relevant resistance to all beta-lactams (2,3,7,8,14,18,21,23).Hybridization of the probes with the chromosomal DNA target, immobilized on a nitrocellulose filter, has shown that the presence of the PBP 2a gene in both Staphylococcus aureus and coagulase-negative staphylococci correlates in almost every instance with the phenotypic expression of methicillin resistance detected by in vitro susceptibility tests (1,12).These studies have provided the basis for introducing tests for genetic identification of methicillin resistance in clinical microbiology laboratories. These tests may help to overcome the numerous problems posed by the well-known heterogeneous expression of methicillin resistance, which makes routine susceptibility tests unreliable in detecting this resistance (3,4,15,17,19 Lysis procedure. A number of colonies obtained after overnight growth on tryptic soy agar (TSA; Difco) or mannitol salt agar (MSA; Difco) were suspended in 1 ml of water to a final density of 1 unit at an optical density at 600 nm, which corresponded to 109 CFU/ml. The suspension was centrifuged, and the pellet was resuspended in 100 p1l of 50 mM Tris (pH 8) containing 50 mM NaCl, 25% sucrose, 10 ,ug of lysostaphin (Sigma Chemical Co., St. Louis, Mo.), and 100 pug of lysozyme (Sigma). After incubation at 37°C for 60

A working scheme developed in our laboratory for identification (by species group and species) of coagulasenegative staphylococci (CNS) was evaluated with 201 consecutive isolates and then validated by using the reference method of Kloos and Schleifer (W. E. Kloos and K. H. Schleifer, J. Clin. Microbiol. 1: [82][83][84][85][86][87][88] 1975). This five-test simple scheme (referred to here as the simple scheme) combines the novobiocin susceptibility test with tests for urease, pyrrolidonyl arylamidase, ornithine decarboxylase, and aerobic acid from mannose. The addition of one or two tests within a particular species group could then positively identify the isolate. Two commercial systems, Staph-Zym (Rosco) and API-Staph (bioMérieux), along with results obtained by using Rosco diagnostic tablets (nongrowth tests), were also compared with the reference method. One isolate could not be identified even by the reference method. Of the remaining 200 strains, 191 (95.5%) strains were correctly identified with Staph-Zym and 171 strains (85.5%) were correctly identified with API-Staph. The most frequent clinical CNS species isolated were Staphylococcus epidermidis (50.5%), S. haemolyticus (18.5%), S. saprophyticus subsp. saprophyticus (16.0%), S. lugdunensis (6.0%), and S. warneri (2.5%). The simple scheme validated with the reference method has demonstrated an excellent correlation in the identification of the three most frequent species isolated: S. epidermidis, S. haemolyticus, and S. saprophyticus subsp. saprophyticus. With the simple scheme, identification of CNS was possible within 24 h after the enzymatic tests were used, whereas up to 72 h is necessary for the growth tests. This methodology would be very useful in any clinical microbiology laboratory for the presumptive identification of CNS species groups and species.Many studies have been initiated since 1958 as a result of the growing recognition that coagulase-negative staphylococci (CNS) are clinically important (28) in an attempt to classify these organisms (1, 21). In the 1970s, W. E. Kloos and K. H. Schleifer determined the natural relationships of CNS based on systematic studies that allowed these researchers to resolve and characterize different CNS species (14). Species were identified based on an ensemble of morphological, physiological, and biochemical characteristics, antibiotic susceptibility patterns, and cell wall composition.In the last decade, molecular studies contributed to a notable progress in the classification of staphylococci and in the development of methods for identifying them at the genus, species, subspecies, and strain levels (5). Although Staphylococcus epidermidis accounts for most CNS infections, many other species have been identified in association with human infections (3, 27, 29). The importance of CNS as major nosocomial pathogens is mainly associated with prosthetic and indwelling devices such as prosthetic joints, heart valves, pacemaker implants, ventricular-peritoneal shunts, and peritoneal dialysis catheters and with s...

The antibiotic susceptibility rates of 363 clinical Bacteroides fragilis group isolates collected from 17 centers in Argentina during the period from 2006 to 2009 were as follows: piperacillin-tazobactam, 99%; ampicillin-sulbactam, 92%; cefoxitin, 72%; tigecycline, 100%; moxifloxacin, 91%; and clindamycin, 52%. No metronidazole resistance was detected in these isolates during this time period. Resistance to imipenem, doripenem, and ertapenem was observed in 1.1%, 1.6%, and 2.3% of B. fragilis group strains, respectively. B. fragilis species showed a resistance profile of 1.5% to imipenem, 1.9% to doripenem, and 2.4% to ertapenem. This is the first report of carbapenem resistance in Argentina. The cfiA gene was present in 8 out of 23 isolates, all of them belonging to the B. fragilis species and displaying reduced susceptibility or resistance to carbapenems (MICs > 4 g/ml). Three out of eight cfiA-positive isolates were fully resistant to carbapenems, while 5 out of 8 isolates showed low-level resistance (MICs, 4 to 8 g/ml). The inhibition by EDTA was a good predictor of the presence of metallo-␤-lactamases in the fully resistant B. fragilis strains, but discrepant results were observed for low-level resistant isolates. B. fragilis was more susceptible to antimicrobial agents than other Bacteroides species. Bacteroides vulgatus species was the most resistant to ampicillin-sulbactam and piperacillin-tazobactam, and B. thetaiotaomicron/ovatus strains showed the highest level of resistance to carbapenems, with an unknown resistance mechanism. B. vulgatus and the uncommon non-Bacteroides fragilis species were the most resistant to moxifloxacin, showing an overall resistance rate of 15.1%.

The efflux mechanism seems to be an important mechanism to confer resistance to antibiotics and biocides through MDR pumps. It was observed that several qac genes coexist in some of the isolates and seem to act simultaneously in the removal of different compounds out of the bacterial cell. The qac genes are horizontally spread among different clones.