Double Disk Diffusion Test Research Articles

Prevalence of extended-spectrum b-Lactamases producing Aeromonas hydrophila isolated from stool samples collected in the Limpopo province, South Africa

Aeromonas producing extended-spectrum s-lactamases (ESBLs) have been reported in many countries, but there is no information on the prevalence of ESBL-producing clinical Aeromonas in South African. A total of 230 isolates of Aeromonas hydrophila were isolated from 660 stool samples and 709 water samples collected in different municipalities in Limpopo province, South Africa over a period of three month. Isolates were screened for the production of ESBLs by the double disk diffusion test and for AmpC production by assessing resistance to cefoxitin. blaSHV,blaTEM , blaCTX-M and blaCMY-2 were isolated from all ESBL-positive and cefoxitin-resistant isolates. Only 21 isolates were found to be ESBL producers. All 21 isolates were screened for the production of blaSHV, blaTEM, blaCTX-M and blaCMY-2 ESBLs. Only (1/21) was found to produce blaTEM 63 and blaCMY-2. All transconjugants were resistant to amoxicillin, piperacillin, the cephalosporins and aztreonam but remainedsusceptible to cefoxitin and imipenem. Crude extracts of s-lactamase-producingtransconjugants were able to reduce the diameters of inhibition zones around disks containing penicillin, but had no effect on such zones around cefoxitin, imipenem and amoxicillin-clavulanate disks. In conclusion, the occurrence of ESBLs in A .hydrophila emphasizes the importance of constant surveillance of clinical isolates to determine the prevalence of antibiotic resistance genes. Key words: Extended-spectrum b-lactamases- Aeromonas hydrophilia , antibiotics , resistance.

Transmission of a Klebsiella pneumoniae clone harbouring genes for CTX-M-15-like and SHV-112 enzymes in a neonatal intensive care unit of a Kuwaiti hospital

The spread of antibiotic-resistant bacteria has become a large problem in most countries including Kuwait. This antibiotic resistance is usually due to the production of extended-spectrum beta-lactamase (ESBL) enzymes such as SHV, TEM and CTX-M. This study reports the emergence and spread of an ESBL-producing Klebsiella pneumoniae clone in a neonatal intensive care unit (NICU) in a Kuwaiti hospital. Eight ESBL-producing K. pneumoniae isolates were from blood cultures of seven neonates, and two were from the fingers of two healthcare workers in a NICU in Al Jahra Hospital, Kuwait. All isolates were obtained in February-March 2006, except for one, which was obtained in August 2005. Identification of the bacteria was based on traditional bacteriological and biochemical tests using the Vitek system. Antibiotic susceptibility was tested by the disc diffusion method using 16 different antibiotics. ESBLs were detected using disc approximation and double-disc synergy methods and confirmed as ESBLs using Etest. PCR and DNA sequencing were performed to determine the genotypes and mutations in the beta-lactamase genes (blaTEM, blaSHV and blaCTX-M). Genetic relatedness was determined by PFGE. All isolates were confirmed to have ESBLs by the Vitek system, disc approximation test, double-disc diffusion test and Etest, being resistant to cefotaxime, ceftazidime, cefepime, gentamicin, tobramycin and ciprofloxacin but susceptible to tetracycline and trimethoprim-sulfamethoxazole. Molecular studies showed the isolates to have TEM-1 beta-lactamase, a CTX-M-15-like ESBL and the newly discovered SHV-112 ESBL. PFGE showed that all isolates had identical banding patterns. The results indicate that a single clone of ESBL-producing K. pneumoniae caused bloodstream infections among babies in a NICU of a Kuwaiti hospital, and may have emerged at least 5 years ago. This clone was also present on the hands of healthcare workers, suggesting that they may have been involved in its transmission. Further studies are recommended to determine whether this clone is also spreading in other Kuwaiti hospitals.

Can we rely on one laboratory test in detection of extended-spectrum β-lactamases among Enterobacteriaceae? An evaluation of the Vitek 2 system and comparison with four other detection methods in Kuwait

Background and Aims:Clinical hospitals need to correctly identify extended spectrum β-lactamase (ESBL)-producing bacteria in infected patients to correctly treat the patient and avoid spreading antibiotic resistance. Kuwaiti hospitals use one...

Prevalence of erythromycin and clindamycin resistance among clinical isolates of the Streptococcus anginosus group in Germany

Members of the Streptococcus anginosus group (SAG) are frequently involved in pyogenic infections in humans. In the present study, the antimicrobial susceptibility of 141 clinical SAG isolates to six antimicrobial agents was analysed by agar dilution. All isolates were susceptible to penicillin, cefotaxime and vancomycin. However, 12.8 % displayed increased MIC values (0.12 mg l(-1)) for penicillin. Resistance to erythromycin was detected in eight (5.7 %) isolates. Characterization of the erythromycin-resistant isolates with the double-disc diffusion test revealed Macrolide-Lincosamide-Streptogramin(B) and M-type resistance in six and two isolates, respectively. The erythromycin-resistant isolates were further characterized by PCR for the resistance genes ermA, ermB and mefA. Resistance and intermediate resistance to ciprofloxacin were detected in two and six isolates, respectively. Molecular typing by PFGE revealed a high genetic heterogeneity among the SAG isolates and no evidence for a clonal relationship between the erythromycin-resistant isolates. Our data show that resistance to erythromycin, clindamycin and ciprofloxacin has emerged among SAG isolates in Germany. The implications of these findings for susceptibility testing and antimicrobial therapy of SAG infections are discussed.

T Types,emmGenotypes and Antibiotic Resistance ofStreptococcus pyogenesIsolated from School Children in Jinju, 2006

Background: Streptococcus pyogenes is the most common cause of bacterial pharyngitis. T antigens and emm genotypes are essential markers for an epidemiological study of S. pyogenes. Macrolide resistance of S. pyogenes is a serious obstracle to successfully treating a sore throat. Methods: One-hundred forty-seven strains of S. pyogenes isolated from healthy school children in 2006 were subjected to T typing and emm genotyping. A disk diffusion method was applied for several antibiotics. A double disk diffusion test was performed to evaluate the phenotype distribution of macrolide resistance. Results: Among T antigens and emm genotypes, T11 (19.7%) and emm78 (16.7%), respectively, were the most common in 2006. Both T5/27/44 (2.3%) and emm44/61 (9.1%) declined to a great extent from about 29% in 2004. The rate of resistance to antibiotics were 11.6% to erythromycin, 4.8% to clindamycin, 21.8% to tetracycline, and 7.5% to ofloxacin. M and cMLSB phenotypes were 52.9% and 41.2% respectively. Conclusion: T typing and emm genotyping proved a dynamic change in their distribution in 2006 compared to the results of 2004. Erythromycin and clindamycin resistance remained low as in 2004, whereas ofloxacin resistance increased slightly. M and cMLSB phenotypes were equivalent in 2006, whereas cMLSB was predominant in 2004. (Korean J Clin Microbiol 2009;12:6-10)

Extended Spectrumβ-Lactamases among Gram-Negative Bacterial Isolates from Clinical Specimens in Three Major Hospitals in Northern Jordan

Background and Objectives. Extended spectrum β-lactamase (ESBL) production is increasing all over the world, and organisms other than E. coli and K. pneumoniae are acquiring this character. ESBL production is detectable by automation, E-test, double disk diffusion (DDD), and PCR. This study aimed to determine the prevalence of ESBL production among clinical isolates of gram-negative rods, and to evaluate the effectiveness of augmentation of clavunate with Cefotaxime, Ceftazoxime, Aztreonam, Ceftriaxone, and Cefpodoxime in detecting ESBL production. Methods. 472 clinical gram-negative isolates identified by standard methods were tested for ESBL-production by (DDD) method using six cephalosporins and amoxicillin-clavulinate discs. Results. 108/472 (22.9%) of the isolates were ESBL producers, and were prevalent in tertiary care hospitals. 88.2% of E. cloacae, 71.4% of K. pneumoniae, 28.6% of K. oxytoca, 12.5% of C. freundii, 11.1% of A. calcoacceticus, and 10.8% of E. coli were ESBL producers. The DDD test demonstrated some variations in the efficacy of the different cephalosporins in detecting all the ESBL producers. The inclusion of ceftizoxime discs increased the efficacy of the test. It is concluded that ESBL-producing bacteria were prevalent among our hospitalized patients, and involved genera other than Klebsiella and Escherichia, and the inclusion of ceftizoxime increased the efficacy of ESBL detection by the DDD test.

Hydrolysis of cefazolin by enzymes produced by Pseudomonas aeruginosa after exposure to ceftazidime in vitro

Sometimes resistance of Pseudomonas aeruginosa (Ps. aeruginosa) is developed during antibiotic treatment, in spite of the initial susceptibility in vitro. The aim of this study was to use an in vitro model for the study of the development of resistant strains of Ps. aeruginosa after a short exposure to ceftazidime, and to study the hydrolysing capacity of beta-lactamases produced by the resistant strains. Among 563 clinical strains of Ps. aeruginosa, 37 multisensitive strains were collected for the study. After being identified, strains with simultaneous sensitivity to 5 expanded spectrum cephalosporins were chosen. For each strain, the minimal inhibitory concentration (MIC) of the 5 expanded spectrum cephalosporins was determined, and the production of extended spectrum beta-lactamases (ESBL) was excluded by the double-disc synergy diffusion test. Strains non producing ESBL were cultivated in concentrations of ceftazidime equal to MICx2 and MICx4. After 24 hours of culture, the development of resistant strains was estimated and the cephalosporinase activity of the produced beta-lactamases was determined by their ability to hydrolyse cefazolin. Hydrolysis of cefazolin was studied by measuring the change of its absorbance on 272 nm using a Shimadzu 160A spectrophotometer. The hydrolyzing capacity of the enzymes was expressed as the percentage of the antibiotic, which was hydrolysed in 10 sec. A total of 60% and 50% of strains developed resistant strains after exposure to ceftazidime in concentration MICx2 and MICx4, respectively. The hydrolyzing capacity of the original strains was 15-36% while the hydrolyzing capacity of the resistant strains was 10-73%. Totally 64% of the resistant strains expressed higher hydrolyzing capacity than the original strains. Regardless of the susceptibility test results, Ps. aeruginosa presented a high tendency to develop resistant strains after a short exposure to ceftazidime in vitro. In most cases the resistant strains expressed higher cephalosporinase activity than the original strains, suggesting derepression of chromosomal beta-lactamases. Our model offers a simple, inexpensive and rapid method for detecting resistance of Ps. aeruginosa developed due to derepression of beta-lactamases, and for discriminating resistant strains with derepressed beta-lactamases from strains that developed other mechanisms of resistance.

Genetic Structure Associated withblaOXA-18, Encoding a Clavulanic Acid-Inhibited Extended-Spectrum Oxacillinase

The genetic environment of the bla(OXA-18) gene encoding a peculiar clavulanic acid-inhibitable Ambler class D extended-spectrum beta-lactamase was determined from the prototype OXA-18-producing Pseudomonas aeruginosa MUS clinical isolate. An 8.2-kb genomic DNA fragment containing bla(OXA-18) was cloned from P. aeruginosa MUS. Although most oxacillinases are located in integrons, bla(OXA-18) lacked gene cassette-specific features. It was bracketed by two duplicated sequences containing ISCR19, a novel insertion sequence of the ISCR family of mobile elements; DeltaintI1, a truncated integrase gene; and a truncated Deltaaac6'-Ib gene cassette. It is likely that ISCR19 was at the origin of the bla(OXA-18) gene mobilization by a rolling-circle transposition event followed by homologous recombination. Furthermore, analysis of the cloned genomic DNA fragment revealed the presence of the integron-containing bla(OXA-20) gene. Concomitantly, three P. aeruginosa clinical isolates, displaying a synergy image as determined by double-disk diffusion tests on cloxacillin-containing plates, were isolated from three patients hospitalized in different wards over a 9-month period at the Saint-Luc University hospital (Brussels, Belgium). These isolates were positive by PCR for bla(OXA-18) and bla(OXA-20) genes, genetically related to P. aeruginosa MUS as determined by pulsed-field gel electrophoresis, and carried the same bla(OXA-18)/bla(OXA-20)-associated genetic structures. This report characterized the genetic elements likely at the origin of bla(OXA-18) gene mobilization in P. aeruginosa and suggests the spread of oxacillin-type extended-spectrum beta-lactamases in P. aeruginosa at the Saint-Luc University hospital of Brussels, Belgium.

In vitro Antimicrobial Activity of Ampicillin-Ceftriaxone and Ampicillin-Ertapenem Combinations Against Clinical Isolates of Enterococcus faecalis with High Levels of Aminoglycoside Resistance.

This paper reports on the in vitro antimicrobial activity of ampicillin-ceftriaxone and ampicillin-ertapenem combinations against five strains of E. faecalis with high-level aminoglycoside resistance recovered from blood of septicemic patients. Double disk diffusion test and time killing curves were used. A bacteriostatic synergistic effect between ampicillin and ceftriaxone was detected using the disk diffusion assay for three of the five enterococcal strains studied. With the same three isolates enhanced bactericidal activity was also observed using time killing experiments. Overall, for these three strains, after 24 hr of contact, a decrease ≥ 2 log10 from the initial bacterial inoculum was registered with most ampicillin-ceftriaxone combinations, reaching with some of them a colony reduction ≥ 3 log10. This bactericidal interaction was negatively influenced increasing the bacterial inoculum. In all five isolates neither a bacteriostatic nor a bactericidal cooperation was observed for ampicillin combined with 2 mg/l of ertapenem.This investigation broadened the evidence of antimicrobial synergism in vitro between ampicillin and ceftriaxone in selected strains of Enterococcus faecalis with high-level aminoglycoside resistance.

Macrolide–lincosamide–streptogramin B resistance phenotypes in clinical staphylococcal isolates

The prevalence of macrolide–lincosamide–streptogramin B (MLS B) resistance as well as the MLS B resistance phenotypes were investigated by the double-disk diffusion test among 532 clinical staphylococci isolates in a Turkish university hospital. The activity of other antimicrobials, including trimethoprim/sulfamethoxazole, telithromycin, quinupristin/dalfopristin, linezolid, gentamicin, chloramphenicol, ciprofloxacin and vancomycin, was also evaluated. Of 532 isolates, 38.5% were resistant to MLS B antibiotics; 63.9% of the resistant isolates exhibited a constitutive phenotype (cMLS B) whereas 36.1% expressed an inducible resistance phenotype (iMLS B). MLS B resistance was more prevalent among coagulase-negative staphylococci (CoNS) strains. Oxacillin-resistant strains exhibited significantly higher MLS B resistance rates compared with oxacillin-susceptible strains ( P < 0.0001). The most frequently detected resistance phenotype among the total staphylococcal isolates was the constitutive type and this phenotype was more frequently encountered among oxacillin-resistant strains. With the exception of the fully active agents such as vancomycin, linezolid and quinupristin/dalfopristin, the most effective antibiotics were telithromycin and chloramphenicol among all isolates. Susceptibility rates to other antibiotics tested were higher among isolates without MLS B resistance than isolates with MLS B resistance. The detection of a considerable rate (43.5%) of iMLS B resistance among erythromycin-resistant/clindamycin-susceptible strains suggests that the true percentage of clindamycin resistance may be underestimated if testing for inducible resistance is not performed.

Distributions of Macrolide-Lincosamide-Streptogramin B Resistance Phenotypes in Clinical Isolates of Staphylococi

Background: Increased resistance rates to macrolide-lincosamide-streptogramin B (MLSB) antibiotics among clinical isolates of staphylococci are considered as a consequence of an expanded use of these antibiotics in the treatment of Gram-positive infections. The proportion of MLSB resistance phenotypes of staphylococci is quite different by geographical variations and study periods. The aim of the present study was to determine the distribution of MLSB resistance phenotypes among clinical isolates of staphylococci in a university hospital. Methods: The MLSB resistance phenotypes of clinical isolates of staphylococci were investigated by the double-disk diffusion test using erythromycin and clindamycin disks. Results: Of 7,916 isolates, 55.7% exhibited a constitutive resistance phenotype (cMLSB) whereas 8.1% expressed an inducible resistance phenotype (iMLSB). Among 3,419 coagulase-negative staphylococci (CNS), 32.6% and 10.0% exhibited cMLSB and iMLSB resistance phenotypes, respectively. Of 4,497 Staphylococcus aureus isolates, 73.1% and 6.8% were cMLSB and iMLSB resistance phenotypes, respectively. cMLSB was detected among 90.2% of methicillin-resistant S. aureus (MRSA), 46.5% of methicillin-resistant CNS (MRCNS), 3.2% of methicillin-susceptible CNS (MSCNS), and 2.2% of methicillin-susceptible S. aureus (MSSA). iMLSB was detected among 16.5% of MSSA, 11.5% of MRCNS, 6.7% of MSCNS, and 4.4% of MRSA. Conclusion: MLSB resistance was more prevalent among S. aureus isolates than CNS strains. Although cMLSB was the most frequently detected resistance phenotype among the total staphylococcal isolates, methicillin-susceptible strains exhibited somewhat higher iMLSB resistance rates compared with methicillin-resistant strains. (Korean J Clin Microbiol 2008; 11:78-83)

Nosocomial outbreak of OXA-18-producing Pseudomonas aeruginosa in Tunisia

Following systematic screening for ceftazidime-resistant (CAZ-R) Pseudomonas aeruginosa, 24 isolates producing extended-spectrum beta-lactamase (ESBL) were recovered during a 24-month period at the National Bone Marrow Transplant Centre of Tunisia. These isolates were from seven immunocompromised patients and from environmental swabs. ESBLs inhibited by clavulanic acid were detected by double-disk diffusion tests. Isoelectric focusing revealed that these isolates produced two to four beta-lactamases with pIs of 5.5, 6.1, 6.4, 7.6 or 8.2, and PCR detected the presence of bla(OXA-18), bla(SHV) and bla(TEM) genes in 24, 21 and two isolates, respectively. Pulsed-field gel electrophoresis defined two dominant genotypic groups: group A (16 isolates) and group B (four isolates). Sequencing of PCR products from representative isolates identified the bla(OXA-18) gene and revealed nucleotide sequences belonging to the bla(SHV-1) and bla(TEM-1) genes. Isolates producing OXA-18 belonged to genomic group A and were isolated from four immunocompromised patients in the haematology and graft units, and from two wash-basins in the graft unit. No immunocompromised patient harboured the clonal epidemic strain upon admission. This is the first report of the OXA-18-type ESBL in P. aeruginosa in Tunisia, and the first description of an outbreak caused by an OXA-18-producing strain of P. aeruginosa.

Comparative evaluation of VITEK 2® for antimicrobial susceptibility testing of group B Streptococcus

Intrapartum antibiotic prophylaxis is recommended to prevent neonatal group B streptococcal (GBS) disease in colonized women, and penicillin or aminopenicillin constitute the first-line antibiotics. Most isolates are resistant to tetracycline, and resistance to macrolide-lincosamide-streptogramin (MLS) antibiotics is increasing. Therefore, laboratory testing for MLS resistance in GBS is now recommended for penicillin-allergic patients. The aim of this study was to compare the antimicrobial susceptibility of GBS as determined by the VITEK 2 system (bioMérieux, Marcy l'Etoile, France), agar diffusion methods and PCR-genotypic detection of resistance genes. One hundred and ten unrelated selected GBS clinical isolates were studied. The antibiotics tested (VITEK 2 and agar diffusion method) were benzylpenicillin, ampicillin, erythromycin, clindamycin, co-trimoxazole, tetracycline, kanamycin, streptomycin and vancomycin. A standardized double-disc (DD) diffusion test was performed for MLS antibiotics. Genotypic characterization of tetracycline, MLS and aminoglycoside resistance genes was performed by PCR. All strains were susceptible to benzylpenicillin, ampicillin and vancomycin [category agreement (CA) between VITEK 2 and the diffusion method was 100%]. Ninety-five (86%) strains were resistant to tetracycline (CA was 98.9%). Eighty-one strains (73.6%) harboured an MLS resistance phenotype; 50 (61.8%) an MLS(B)-constitutive phenotype, 25 (30.8%) an MLS(B)-inducible phenotype and 6 (7.4%) an M phenotype. The agreement between data of VITEK 2 and the DD diffusion test for the detection of MLS(B)-constitutive, MLS(B)-inducible and M phenotype isolates was 76%, 36% and 100%, respectively. Almost all discrepancies were due to failure to detect erythromycin resistance by VITEK 2. VITEK 2 allows accurate determination of GBS susceptibility for the majority of antibiotics, but has to be improved for erythromycin. Thus, the DD diffusion test remains the most simple and reliable method for macrolide resistance detection among this streptococcal species.

Detection and prevalence of inducible clindamycin resistance in staphylococci

Staphylococcus aureus and coagulase-negative staphylococci (CNS) are recognized as causing nosocomial and community-acquired infections in every region of the world. The resistance to antimicrobial agents among staphylococci is an increasing problem. Clindamycin (CL) is considered to be one of the alternative agents in these infections. This study demonstrates a simple, reliable method (double-disc diffusion test) for detecting inducible resistance to CL in erythromycin-resistance (ER-R) isolates of S. aureus and CNS. A total of 883 (52.3%) isolates of S. aureus and 804 (47.7%) isolates of CNS were selected from recent (2003-2005) clinical isolates recovered in the laboratory of the authors; duplicate isolates were not included. A total of 214 (12.6%) S. aureus and 308 (18.3%) CNS isolates were selected based on ER-R and CL sensitivity using standard National Committee for Clinical Laboratory Standards disc diffusion testing. A total of 1687 staphylococcal isolates were included, consisting of 27.5% meticillin-resistant S. aureus, 24.8% meticillin-sensitive S. aureus, 36.1% meticillin-resistant CNS and 11.6% meticillin-sensitive CNS isolates: 30.9% of staphylococcal isolates (214 S. aureus and 308 CNS) that were erythromycin resistant and CL sensitive were tested for inducible resistance using the D-test. A D-shaped zone around the CL was observed for 70.9% of staphylococcal isolates (81.8% of S. aureus isolates and 63.3% of CNS isolates) with an ER-R and a clindamycin-sensitive (CL-S) phenotype. The organism was positive for inducible clindamycin resistance (CL-R). There was a 21.9% level of inducible macrolide-lincosamide-streptogramin B resistance phenotype among all the staphylococcal isolates. When the S. aureus and CNS strains among all the staphylococcal isolates were compared statistically, inducible CL-R in CNS strains was determined to be 23% more positive (P=0.028, odds ratio 0.77, 95% confidence interval 0.61-0.98). When a statistical comparison was performed among ER-R but CL-S staphylococcal isolates inducible CL-R in S. aureus strains was determined to be 2.6 times more positive (P=0.000, odds ratio 2.6, 95% confidence interval 1.68-4.04). A simple, reliable method of detecting inducible resistance to CL in ER-R isolates of S. aureus and CNS is described. Clinical microbiology laboratories should use the double-disc diffusion test as standard practice with all ER-R staphylococci. CL should not be used in patients with infections caused by inducibly resistant staphylococcal isolates. Therapeutic failures may thus be avoided.

Detección de resistencia inducible a clindamicina en aislados cutáneos de Staphylococcus spp. por métodos fenotípicos y genotípicos

Resistance to macrolides, lincosamides and type B streptogramins (MLSB) in Staphylococcus isolates can be due to several mechanisms. The most important are an active efflux mechanism (MSB phenotype) and ribosomal target modification (MLSB phenotype); this latter mechanism confers resistance to all three groups of antimicrobials (MLSB resistance). Expression of MLSB resistance can be constitutive (cMLSB) or inducible (iMLSB). A group of 117 erythromycin-resistant Staphylococcus spp. clinical isolates from cutaneous samples were selected from 536 recent clinical isolates of this microorganism. Resistance phenotypes were determined by the double disk diffusion test. Presence of the ermA, ermC, ermB and msrA genes was detected by real time PCR. The MSB phenotype was the most common, comprising 11.2% (7.2% in S. aureus and 23% in CoNS) of the erythromycin-resistant strains. The rate of iMLSB resistance was significantly higher, 7.4% (5.2% in S. aureus and 14% in CoNS), than the rate of cMLSB resistance, 3.2% (1.7% in S. aureus and 7.4% in CoNS). The msrA gene was present in all isolates with the MSB phenotype, and the ermC gene was the most common among clindamycin-resistant strains with the MLSB phenotype (constitutive or inducible). The good correlation between the phenotypic (disk-diffusion) and genotypic (real time PCR) methods used allows prediction of the mechanisms of erythromycin and clindamycin resistance, provides insight into the epidemiological differences in their distribution, and is an aid to selecting the most appropriate antimicrobial therapy.

Prevalence of multiresistant Gram‐negative organisms in a surgical hospital in Ho Chi Minh Cit, Vietnam

To determine resistance patterns of multiresistant Gram-negative organisms at a surgical hospital in Ho Chi Minh City, Vietnam, in order to guide appropriate antibiotic prescribing and improve infection control procedures. All samples sent in for microbiological analysis over a 3-month period were included. A resource neutral double disc-diffusion test was introduced to detect the presence of extended-spectrum beta-lactamase (ESBL) production. We obtained 350 bacterial isolates from clinical specimens; 87.4% were Gram-negative bacteria (GNB). Of these, 88.9% were Enterobacteriaceae, of which 14.7% produced ESBL. Fifteen (37.5%) of these were isolated within 48 h of admission. Resistance to gentamicin and ciprofloxacin occurred in 70.0% and 72.5% of those organisms that produced ESBL and in 39.5% and 38.7% of those that did not. Resistance to third-generation cephalosporins was common: 36.7% of all GNB were resistant to ceftriaxone, 34.0% to cefotaxime, 19.6% to ceftazidime and 36.7% to cefoperazone. Multiresistant Gram-negative organisms are common and pose a challenge to antibiotic therapy. Successful implementation of a simple test to detect ESBL production allowed reporting of these organisms, appropriate antibiotic prescribing and infection control interventions. Development of antibiotic-prescribing guidelines must take into account these resistance patterns.

Comparisons of Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) and Hospital-Associated MSRA Infections in Sacramento, California

Methicillin-resistant Staphylococcus aureus (MRSA) has long been a common pathogen in healthcare facilities, but in the past decade, it has emerged as a problematic pathogen in the community setting as well. A retrospective case series study of patients from whom MRSA was isolated from December 1, 2003, through May 31, 2004, was conducted at the University of California, Davis, Medical Center. Patient data were collected from electronic medical records and traditional chart reviews to determine whether MRSA acquisition was likely to have been in the community or in the hospital. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed for all confirmed isolates. Skin and soft tissue were the most common infection sites for all MRSA patients. Among the 283 MRSA infections, 127 (44.9%) were defined as community-associated (CA)-MRSA. Ninety-six percent of the CA-MRSA isolates were susceptible to clindamycin. Double-disk diffusion tests were performed to examine inducible clindamycin resistance by erythromycin induction on both CA and hospital-associated (HA) clindamycin-susceptible and erythromycin-resistant isolates. Ten percent (17 of 183) were positive. Most CA-MRSA isolates were identified by PFGE as a unique strain, genotype USA300, which was not genetically related to the predominant genotype, USA100, in the HA-MRSA isolates. Injecting drug users accounted for 49% of CA-MRSA infections but only 19% of the HA-MRSA infections (odds ratio, 4.2; 95% confidence interval, 2.4 to 7.4). Our study shows that a single clone of CA-MRSA accounts for the majority of infections. This strain originated in the community and is not related to MRSA strains from healthcare settings. Injecting drug users could be a major reservoir for CA-MRSA transmission.

Characterization of extended-spectrum β-lactamase (ESBL)-producing Kuwait and UK strains identified by the Vitek system, and subsequent comparison of the Vitek system with other commercial ESBL-testing systems using these strains

Two hundred and fifty-one unique patient isolates of Klebsiella pneumoniae (123), Escherichia coli (114), Klebsiella oxytoca (7), Enterobacter cloacae (5) and Citrobacter freundii (2), flagged as extended-spectrum beta-lactamase (ESBL) positive by the Vitek system (GNS-526 card), were collected. These strains were isolated from a variety of clinical specimens submitted to the clinical bacteriology laboratories of the Royal Infirmary of Edinburgh (RIE), Edinburgh, UK (and associated GP practices), Hairmyers Hospital, Glasgow, UK, and the Amiri and Farwania Hospitals, Kuwait. Of the 101 RIE strains tested, 15 E. coli strains were found to be ESBL negative by Etest ESBL strips. On retesting the 15 E. coli strains with the Vitek GNS-532 card, 14 were found to be ESBL negative, despite being originally flagged as ESBL positive. The remaining 236 ESBL-producing strains were also subjected to the double disc-diffusion (DDD) technique for the detection of ESBLs. Of these, two were false negatives by Etest ESBL test strips (using both cefotaxime and ceftazidime strips), and 38 were false negatives by the DDD method. The Etest false-negative ESBL-producing strains of K. pneumoniae were positive by DDD. Technically, the Vitek method was the least demanding method to perform, as it was an integral part of the routine susceptibility test card. Etest strips were reliable, but were the most expensive of all the techniques used. The DDD test, while relatively inexpensive, was technically subjective, and in our hands, seven of the ESBL-positive strains that were confirmed by the other two techniques were not detected. Despite the false-positive ESBL-producing E. coli strains, the Vitek susceptibility card with its integral ESBL test offers the clinical laboratory a valuable and quick option to screen for ESBL-producing Klebsiella spp. and E. coli as part of the routine laboratory methodology.

Increasing Clindamycin Resistance Among Methicillin-Resistant Staphylococcus aureus in 57 Northeast United States Military Treatment Facilities

Increasing community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has led many to use clindamycin for CA-MRSA disease, whereas others suggest caution because of inducible clindamycin resistance and rely on drugs such as trimethoprim-sulfamethoxazole (TMP-SMX). To analyze the change in antibiotic susceptibility pattern of S. aureus isolates from 2001 to 2004 in a closed health care system, a period during which clindamycin and TMP-SMX use increased 99 and 62%, respectively. S. aureus cultures from 57 military hospitals and clinics from 2001-2002 and 2003-2004 were compared for body site and antibiotic sensitivity, particularly the potentially inducible clindamycin-susceptible erythromycin-resistant pattern. A portion was evaluated by a double disk diffusion test (D test). Numbers of S. aureus-positive cultures obtained from "wound" sites rose significantly in both pediatric (138 to 215, P < 0.001) and adult (715 to 972, P < 0.001) isolates, with a rise in MRSA in children (6 of 138 to 60 of 215, P < 0.001) and in adults (161 of 715 to 324 of 972, P < 0.001). Clindamycin resistance increased among pediatric S. aureus isolates (1 of 207 to 13 of 327, P < 0.05), whereas >96% remained TMP-SMX-susceptible. Five methicillin-susceptible S. aureus (MSSA), 2 MRSA of 41 pediatric and 36 MSSA, 43 MRSA of 437 adult S. aureus specimens were D test-positive. In late 2004, most S. aureus from our region are still beta-lactam-susceptible. Most of the MRSA are still susceptible to clindamycin and do not appear to have inducible resistance. We must closely monitor the rates of constitutive and inducible clindamycin resistance as well as consider treatment alternatives that may slow the rate of clindamycin resistance.

Resistencia enzimática a betalactámicos en el género Proteus y evaluación de los fenotipos y genotipos de resistencia a cefalosporinas de tercera y cuarta generación en Proteus mirabilis

The aim of this study was to evaluate betalactam resistance within the genus Proteus and characterize the betalactamases responsible for this resistance. We analyzed 99 strains (87, P. mirabilis; 10 P. vulgaris, and 2, P. penneri) isolated from patients at one University Hospital. Antibiotic susceptibility tests were performed according to NCCLS recommendations. Presence of extended spectrum betalactamases (ESBL) was inferred by both double disk diffusion tests and minimum inhibitory concentration (MIC) of third and fourth generation cephalosporins alone and in the presence of clavulanic acid. Isoelectric points (pI) of the enzymes were estimated by isoelectrofocusing and the presence of the encoding genes was confirmed by polymerase chain reaction (PCR). A broad spectrum betalactamase could be detected in those isolates (28%) resistant to penicillin and first generation cephalosporins while CTX-M-2 enzyme could be detected in P. mirabilis isolates resistant to third and fourth generation cephalosporins (18%). One of the P. vulgaris displayed reduced susceptibility to cefotaxime due to an enzyme of pI 7.4, while resistance to cefotaxime in one P. penneri was related to an enzyme of pI 6.8. Both enzymes were active on cefotaxime (1,000 mg/l) in the iodometric assay. The broad extended spectrum betalactamase within genus Proteus was TEM-1, while CTX-M-2 was the ESBL responsible for the third and fourth generation cephalosporins in P. mirabilis. In P. vulgaris and P. penneri this resistance was associated with the hyperproduction of the chromosomal encoded betalactamase.