Evaluation of discrepancies between oxacillin and cefoxitin susceptibility in coagulase-negative staphylococci

The Clinical and Laboratory Standards Institute (CLSI) recommends testing coagulase-negative staphylococci (CoNS) strains to determine resistance against oxacillin by testing for mecA, PBP2a, or with cefoxitin disk. However, discrepant results of resistance to oxacillin and susceptibility to cefoxitin were found. In this study, we aimed to investigate the oxacillin resistance and cefoxitin susceptibility of CoNS in Taiwan. Of 9,017 strains collected from 2005 to 2010, 131 (1.5%) of the isolates were oxacillin-resistant and cefoxitin-susceptible. Species identification was carried out using the Vitek 2 system or 16S ribosomal RNA sequencing. Oxacillin minimum inhibitory concentrations (MICs) were examined by the agar dilution method. The presence of mecA and the activity of β-lactamase were performed by polymerase chain reaction (PCR) and Cefinase disks, respectively. Overall, 33% (43/129) of the strains carried mecA and 43% (37/86) of mecA-negative isolates tested positive for β-lactamase. The remaining 49 isolates were negative for both mecA and β-lactamase, and were mainly Staphylococcus cohnii ssp. urealyticus and S. saprophyticus (oxacillin MICs 0.5–2 μg/ml) obtained from bloodstream and urinary tract infections. Our study suggests that incorrect reporting can be found in CoNS using cefoxitin disk alone to determine the susceptibility to oxacillin, and the strains should be further tested for oxacillin MICs and detection of the mecA gene or β-lactamase activity.     

Use of the Polymerase Chain Reaction for Rapid Detection of High-Level Mupirocin Resistance in Staphylococci

 The minimum inhibitory concentrations (MICs) of mupirocin were determined by the E test (AB Biodisk, Sweden) and the agar dilution method for 107 staphylococci. The organisms consisted of 34 coagulase-negative staphylococci and 73 methicillin-resistant Staphylococcus aureus. Polymerase chain reaction (PCR) primers designed to amplify a 456 bp region of the plasmid-borne isoleucyl tRNA synthetase gene (ileS–2), responsible for high-level mupirocin resistance in staphylococci, were used on DNA preparations from these isolates. Isolates with high-level mupirocin resistance due to the ileS–2 gene should be PCR positive. There was close correlation between the E test and agar dilution MIC values, with only two strains differing by more than two serial dilutions. Most (51 of 54 strains) of the high-level resistant strains (MIC>256 μg/ml) were resistant to the highest concentration of mupirocin tested (1024 μg/ml). PCR correctly classified all but four (96%) of the isolates in accordance with the results of agar dilution. All four isolates that gave discrepant results were methicillin-resistant Staphylococcus aureus. Two of these were PCR positive, yet the MIC of mupirocin for these strains was <0.06 μg/ml; on prolonged incubation they produced halos within the inhibition zone on agar diffusion testing, suggesting that the phenotypic results may have been erroneous. One of 54 isolates for which the MIC exceeded 256 μg/ml was PCR negative when tested by the original methodology, but a 456 bp product was produced when retested using a lowered annealing temperature. One isolate for which the MIC of mupirocin was 16 μg/ml by agar dilution and 8 μg/ml by the E test was positive by PCR. PCR of the ileS–2 gene is a useful, rapid method for detecting high-level mupirocin resistance in staphylococci.     

Comparative Activity of Eighteen Antimicrobial Agents against Anaerobic Bacteria Isolated in South Africa

 The in vitro activity of 18 antimicrobial agents was determined against 378 anaerobic bacteria isolated in Bloemfontein, South Africa, during 1996/97. Against the gram-positive isolates, MICs of penicillin and cefoxitin were >0.5 μg/ml and >16 μg/ml, respectively, for five and three strains of non-perfringens Clostridium spp. Seventeen Peptostreptococcus anaerobius strains were resistant to penicillin (MIC≥2 μg/ml). All gram-positive anaerobes tested except one Peptostreptococcus sp. and one Clostridium sp. were susceptible to dalfopristin-quinupristin (MICs≤1 μg/ml). The carbapenems exhibited excellent activity against the gram-positive isolates and were effective against most gram-negative anaerobes, with the exception of the fusobacteria. Only seven strains exhibited decreased susceptibility to trovafloxacin (MICs>2 μg/ml). In mixed anaerobic/aerobic infections, carbapenems and the fourth-generation quinolone trovafloxacin were the agents most suitable for us as broad-spectrum monotherapy.     

Variations of agar screen tests for detection of methicillin resistance in staphylococci: focus on cefoxitin

Members of the genus Staphylococcus are among the most important human pathogens, and strains demonstrating resistance to methicillin are an increasing problem worldwide, both within and outside of hospital environments. The objective of this study was to evaluate the use of variations of agar screening tests with cefoxitin and oxacillin to detect methicillin resistance in staphylococcal isolates. The agar screening test with cefoxitin (4 μg/ml) showed 99.4% accuracy for detecting both S. aureus and coagulase-negative staphylococci. The performance of the agar screening test with cefoxitin (4 μg/ml) either equaled or was superior to the other agar screening test variations evaluated and can be used to characterize the presence of the mecA gene among staphylococcal species.     

Minimal inhibitory concentration of isoniazid in isoniazid-resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> isolates from children

The aim of the study presented here was to determine the minimal inhibitory concentration of isoniazid for strains of isoniazid-resistant or multidrug-resistant Mycobacterium tuberculosis isolated from children in the Western Cape Province of South Africa. During the period March 2003–October 2005, 45 INH-resistant M. tuberculosis isolates (21 also rifampicin-resistant) were cultured from children less than 13 years of age. Drug susceptibility testing by the radiometric BACTEC 460 method found 11 isolates resistant at 0.1 μg/ml, 27 resistant at 0.2 μg/ml, and seven resistant at ≥5 μg/ml. Thus, the minimal inhibitory concentration of isoniazid for more than 80% of the isoniazid-resistant strains isolated from children in this study was relatively low and could be exceeded by high-dose (15–20 mg/kg) isoniazid regimens.     

New Colorimetric Microdilution Method for In Vitro Susceptibility Testing of Borrelia burgdorferi Against Antimicrobial Substances

 A newly developed colorimetric microdilution method was used to analyze the activity of 12 antimicrobial agents against nine Borrelia burgdorferi isolates, including all three genospecies pathogenic for humans. In addition, in vitro antimicrobial resistance patterns of Borrelia valaisiana and Borrelia bissettii tick isolates were investigated. The applied test system is based upon color changes that occur in the presence of phenol red and result from the accumulation of nonvolatile acid produced by actively metabolizing spirochetes. After 72 h of incubation, minimal inhibitory concentrations (MICs) were determined from the decrease of absorbance by software-assisted calculation of growth curves. MIC values were lowest for azlocillin (MIC, ≤0.125 μg/ml), ceftriaxone (MIC range, ≤0.015–0.06 μg/ml), and azithromycin (MIC range, ≤0.015–0.06 μg/ml). Whereas tobramycin (MIC range, 8–64 μg/ml) exhibited little activity, spectinomycin (MIC range, 0.25–2 μg/ml) showed in vitro antimicrobial activity against Borrelia burgdorferi. The MICs of penicillin G for Borrelia afzelii isolates were ten times higher than those for Borrelia burgdorferi, Borrelia valaisiana, and Borrelia bissettii isolates (P<0.05) and 100 times higher than those for isolates belonging to the genospecies Borrelia garinii (P<0.05). Further significant differences with respect to the MIC values of the other antimicrobial agents tested were not noted. The colorimetric microdilution method offered the advantages of reliability, reproducibility, and convenience and could handle large numbers of isolates and antibiotics.     

Skin bacteria after chlorhexidine exposure—is there a difference in response to human β-Defensin-3?

We investigated whether exposure to sub-lethal concentrations of chlorhexidine digluconate (CHG) changed the response of five Staphylococcus spp. to human β-Defensin-3 (hBD-3). The change in response for each strain was determined in vitro with time–kill experiments in suspension by comparing the mean log₁₀ reduction caused by hBD-3 at 1.5 and 3 h in exposed and non-exposed bacteria. The identity of staphylococcal species was verified by DNA sequence homology in the gyrA genes in comparison with reference strains. Baseline sub-lethal concentrations allowing visible bacterial growth were between 0.0625 and 0.25 μg/ml. Sub-lethal CHG concentrations increased within 3 days in two isolates. For S. capitis 19/2, CHG-exposed cells were less susceptible to 0.5 μg/ml hBD-3 (log₁₀ reduction 0.78 versus 2.06 at 1.5 h; p < 0.001; t-test). For S. aureus, however, CHG-exposed cells were more susceptible to 1 μg/ml hBD-3. The observed changes between CHG-exposed and non-exposed cells did not indicate a general trend in response to hBD-3. Overall, we found no consistent evidence that 3 days of exposure to CHG changed the response of five Staphylococcus spp. to hBD-3. The use of CHG for skin antisepsis is, based on our data, unlikely to change the natural defence activity of hBD-3.     

Evaluation of a disk diffusion method with cefoxitin (30 μg) for detection of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The emergence of heterogeneous populations of methicillin-resistant Staphylococcus aureus (MRSA) causes major problems in routine screening for MRSA. In heterogeneous MRSA populations, a proportion of bacterial cells show low-level resistance to oxacillin, with minimal inhibitory concentrations (MICs) of oxacillin ranging between 1 and 100 mg/l, while in homogeneous MRSA populations, the MIC of oxacillin for all cells is >100 mg/l. Routine oxacillin disk diffusion tests often fail to detect heterogeneous MRSA populations. In the present study, a recently proposed disk diffusion method that employs a cephamycin antibiotic (cefoxitin 30 g; BD Sensi-disc, Becton Dickinson, Germany) was evaluated using 155 clinical isolates of S. aureus (73 mecA positive and 82 mecA negative). The results were compared with those of other MRSA screening techniques: a disk diffusion test with oxacillin 1 g and cefoxitin 30 g (BD Sensi-disc; Becton Dickinson), an MRSA latex agglutination test (Denka Seiken, Japan), and an oxacillin screen agar test (6 g/ml; Becton Dickinson). Detection of the mecA gene by polymerase chain reaction was considered the gold standard. The performances of the different methods were determined and compared. The results showed that the cefoxitin disk diffusion test is preferable to the oxacillin disk diffusion method for routine screening to detect MRSA.     

Combinations of cefoxitin plus other β-lactams are synergistic in vitro against community associated methicillin-resistant Staphylococcus aureus

In vitro studies demonstrate that oxacillin minimal inhibitory concentrations (MICs) of methicillin-resistant S. aureus (MRSA) strains USA300 and 400 decrease in the presence of cefoxitin. The aim of this study was to characterize the activity of cefoxitin plus β-lactams against a collection of MRSA isolates. We assessed the in vitro antimicrobial activity of a selection of β-lactams alone and together with subinhibitory concentrations of cefoxitin against a collection of MRSA, methicillin-susceptible S. aureus (MSSA), and vancomycin-intermediate S. aureus (VISA) isolates using MICs and time kill assays. For community-associated (CA) MRSA strains USA300 and USA400, MICs of nafcillin, cefazolin, cephalexin, cefuroxime, ceftriaxone and cefotaxime decreased by 8- to 64-times in the presence of 10 μg/ml cefoxitin. In contrast, for hospital-associated (HA) strains COLn, N315, and Mu50, there was no change in any β-lactam MIC in the presence of cefoxitin. When combined with cefoxitin, the cephalexin MIC decreased for eight CA-MRSA and five MSSA sequence types but did not change for seven HA-MRSA sequence types. β-lactam/cefoxitin combinations were synergistic against CA- but not HA-MRSA strains in time kill assays. Cefoxitin combined with a variety of β-lactams enhances their activity against CA-MRSA strains in vitro. Further studies of combination β-lactam therapy may provide insight into β-lactam biology, penicillin binding protein cooperativity, and novel therapeutic strategies against MRSA.     

Comparative in Vitro Activity of Voriconazole and Itraconazole Against Fluconazole-Susceptible and Fluconazole-Resistant Clinical Isolates of Candida Species from Spain

 The in vitro activity of voriconazole was compared with that of itraconazole against 299 fluconazole-susceptible (MIC≤8 μg/ml) and 130 fluconazole-resistant (MIC≥16 μg/ml) clinical isolates of Candida spp. An adaptation of the National Committee for Clinical Laboratory Standards reference method was employed for determination of MICs. Voriconazole showed more potent activity than either fluconazole and itraconazole, even against some Candida albicans, Candida glabrata, and Candida krusei isolates resistant to fluconazole. However, for fluconazole-resistant isolates, the MICs of itraconazole and voriconazole were proportionally higher than for fluconazole-susceptible isolates. These data may indicate cross-resistance.     

In vitro activity of daptomycin against <Emphasis Type="Italic">Staphylococci</Emphasis> isolated from bacteremia and community-onset skin and soft tissue infections in France: data from two nationwide studies

Staphylococci are a leading cause of skin and soft tissue infections (SSTIs) and bacteremia in France, a country with a high prevalence of oxacillin resistance. We evaluated the in vitro activity of daptomycin compared with reference compounds against 445 Staphylococcus aureus and 53 coagulase-negative Staphylococci (CNS) collected during two large nationwide studies performed in 2006 and 2007. The percentage of oxacillin resistance among S. aureus was 13.6% (SSTIs) and 30.7% (bacteremia). Daptomycin showed lower MIC₉₀ levels compared to vancomycin, teicoplanin, and linezolid (0.19 mg/L vs. 2, 1.5, and 1 mg/L, respectively), irrespective of oxacillin susceptibility. Amongst the CNS, 64.2% of the isolates originated from clinical bacteremia were resistant to oxacillin and 24.5% to teicoplanin; all but one Staphylococci were susceptible to daptomycin (MIC = 1.5 mg/l). As with linezolid, daptomycin seems to constitute an alternative option to treat some staphylococcal infections in the French context of high oxacillin resistance prevalence and high glycopeptides MIC.     

In vitro activities of moxifloxacin and tigecycline against bacterial isolates associated with intraabdominal infections at a medical center in Taiwan, 2001–2006

A total of 569 nonduplicate isolates recovered from patients with community-onset or hospital-onset intraabdominal infections (IAIs) from 2001 to 2006 were studied. These included 28 Staphylococcus aureus and 541 Gram-negative isolates (33.6% Escherichia coli, 29.0% Klebsiella pneumoniae, 8.1% Acinetobacter baumannii, and 6.3% Pseudomonas aeruginosa). Minimum inhibitory concentrations (MICs) of the isolates to moxifloxacin, imipenem, and ciprofloxacin were determined using the agar dilution method and to tigecycline using the broth microdilution method. Extended-spectrum β-lactamase (ESBL) producers were found in 15.5% (29 out of 182) of E. coli, 15.3% (24 out of 157) of K. pneumoniae, and 15.4% (2 out of 13) of K. oxytoca isolates. More than 85% of Enterobacteriaceae were susceptible to moxifloxacin, but this percentage was lower among E. coli (78%). The percentage of E. coli (K. pneumoniae) isolates that were not susceptible to moxifloxacin was 6% (0%) in 2001, 39% (17%) in 2003, and 21% (14%) in 2006. Tigecycline exhibited good in vitro activities against all S. aureus and >95% of all Enterobacteriaceae tested. Among the 24 isolates of ESBL-producing K. pneumoniae, 4 had tigecycline MICs ≥2 μg/ml. Eighty percent of A. baumannii isolates exhibited tigecycline MICs of ≤2 μg/ml. This study found that moxifloxacin and tigecycline exhibited good in vitro activity against bacterial isolates causing IAIs.     

COMPARISON OF CEFOXITIN DISC DIFFUSION TEST,OXACILLIN SCREEN AGAR,AND PCR FOR mecA GENE FOR DETECTION OF MRSA

Cefoxitin is a potent inducer of the mecA regulatory system. It is being recommended for detection of methicillin resistance in Staphylococcus aureus (MRSA) when using disk diffusion testing. The aim of our study was to evaluate the efficacy of cefoxitin disc diffusion test to characterize MRSA and compare it with oxacillin agar screening and detection of mecA gene by PCR. Materials and Methods: Fifty strains of S. aureus isolated from clinical samples were used in the study. Routine antibiotic susceptibility testing was performed including oxacillin disk. Oxacillin screen agar plates with 4% NaCl and 6 µg/ml of oxacillin were inoculated and interpreted as per standard guidelines. Cefoxitin disc diffusion test was performed using 30 µg disc and zone sizes were measured. PCR for amplification of the mecA gene was performed. Results: Out of the 50 isolates, 28 were found to be methicillin resistant by oxacillin disc diffusion test, 30 were resistant by oxacillin screen agar method, and 32 were resistant with cefoxitin disc diffusion. For these 32 isolates mecA gene was positive. Conclusion: Results of cefoxitin disc diffusion test is in concordance with the PCR for mecA gene. Thus, the test can be an alternative to PCR for detection of MRSA in resource constraint settings.     

Assessment of the oxacillin disk screening test for determining Penicillin resistance inStreptococcus pneumoniae

The 1 g oxacillin disk diffusion screening test was performed on 1516 recent clinical isolates ofStreptococcus pneumoniae obtained in a 1994–1995 U.S. surveillance study and the results compared to penicillin MICs determined using a standardized broth microdilution method. The oxacillin disk screening method failed to distinguish penicillin-resistant strains from those that were intermediately susceptible. Furthermore, a high percentage (11.1 %) of penicillin-susceptible strains, for which MICs of penicillin were usually 0.06 or 0.03 g/ml, yielded zone diameters of 19 mm with the oxacillin screen test and thus would have been falsely categorized as being resistant to penicillin.     

Heterogeneity of the humoral immune response following <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> bacteremia

Expanding knowledge on the humoral immune response in Staphylococcus aureus-infected patients is a mandatory step in the development of vaccines and immunotherapies. Here, we present novel insights into the antibody responses following S. aureus bacteremia. Fifteen bacteremic patients were followed extensively from diagnosis onwards (median 29 days, range 9–74). S. aureus strains (median 3, range 1–6) and serial serum samples (median 16, range 6–27) were collected. Strains were genotyped by pulsed-field gel electrophoresis (PFGE) and genes encoding 19 staphylococcal proteins were detected by polymerase chain reaction (PCR). The levels of IgG, IgA, and IgM directed to these proteins were determined using bead-based flow cytometry. All strains isolated from individual patients were PFGE-identical. The genes encoding clumping factor (Clf) A, ClfB, and iron-responsive surface-determinant (Isd) A were detected in all isolates. Antigen-specific IgG levels increased more frequently than IgA or IgM levels. In individual patients, different proteins induced an immune response and the dynamics clearly differed. Anti-ClfB, anti-IsdH, and anti-fibronectin-binding protein A IgG levels increased in 7 of 13 adult patients (p < 0.05). The anti-IsdA IgG level increased in 12 patients (initial to peak level: 1.13–10.72 fold; p < 0.01). Peak level was reached 7–37 days after diagnosis. In a bacteremic 5-day-old newborn, antistaphylococcal IgG levels declined from diagnosis onwards. In conclusion, each bacteremic patient develops a unique immune response directed to different staphylococcal proteins. Therefore, vaccines should be based on multiple components. IsdA is immunogenic and, therefore, produced in nearly all bacteremic patients. This suggests that IsdA might be a useful component of a multivalent staphylococcal vaccine.     

Rapid decrease of free vancomycin in dense staphylococcal cultures

Bacterial numbers in broth cultures were determined by bioluminescence assay of intracellular bacterial ATP. Broth MICs for strains of Staphylococcus epidermidis (ATCC 14990 and 35984) and Staphylococcus aureus (ATCC 25923, 29213 and 6538) were determined for cultures with different inocula (10⁵–10⁸ bacteria/ml) after 24 h of incubation in supplemented Mueller–Hinton broth containing vancomycin. All of the tested strains except one were susceptible to methicillin, and all of the strains were susceptible to vancomycin. Free vancomycin concentrations in the broth cultures of all strains were determined with an agar well bioassay after 24 h of incubation. Free vancomycin concentrations and bacterial numbers of ATCC 35984 and ATCC 29213 were also determined after 0.5, 2, 4, and 8 h. In a low inoculum (10⁵ bacteria/ml), the broth MICs were 1–4 μg/ml. In a high inoculum (∼10⁸ bacteria/ml), the broth MICs increased two- to fourfold to 4–8 μg/ml. In dense inocula (∼10⁹–10¹⁰ bacteria/ml), the concentrations of free vancomycin in the broth were reduced, in most cases below the detection limit of the bioassay (≤0.5 μg/ml). This reduction of free vancomycin was fast, occurring in initially dense inocula in less than 30 min. No emergence of resistance was seen. These results show a rapid reduction of free vancomycin in the broth and a simultaneous increase in broth MICs in high inocula, without development of resistance. This indicates that the dosing regimen of vancomycin is of particular importance in staphylococcal infections with dense inocula, e.g. infective endocarditis.     

In vitro activity of tigecycline against methicillin-resistant Staphylococcus aureus, including livestock-associated strains

The in vitro activity of tigecycline was determined using a well-defined collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 202), including 33 livestock-associated strains. Susceptibility testing was performed using the Etest system. Among the 202 MRSA strains, three (1.5%) had a minimum inhibitory concentration (MIC) value for tigecycline greater than 0.5 mg/l, which are considered to be resistant. When these strains were tested using Iso-Sensitest medium, the MICs were substantially lower and no resistance was found. This discrepancy warrants further investigations into the preferred test conditions for tigecycline. In conclusion, tigecycline showed good activity against MRSA strains in vitro.     

Determinants for the Development of Oropharyngeal Colonization or Infection by Fluconazole-Resistant Candida Strains in HIV-Infected Patients

 A point prevalence study to document oral yeast carriage was undertaken. Risk factors for the development of oropharyngeal colonization or infection by fluconazole-resistant Candida strains in HIV-infected patients were investigated with a case-control design. Cases included all patients with fluconazole-resistant strains (MIC≥64 μg/ml), and controls were those with susceptible (MIC≤8 μg/ml) or susceptible-dependent-upon-dose (MIC 16–32 μg/ml) strains. One hundred sixty-eight Candida strains were isolated from 153 (88%) patients, 28 (16%) of whom had oropharyngeal candidiasis. Overall, 19 (12%) of the patients harbored at least one resistant organism (MIC≥64 μg/ml). Among patients with resistant strains, tuberculosis (P<0.001), esophageal candidiasis (P=0.001), clinical thrush (P<0.001), and a CD4+ cell count <200/mm³ (P=0.03) were more frequent. These patients had also been treated more commonly with antituberculous drugs (adjusted odds ratio [OR] 6.13; 95% confidence interval [CI] 2.11–17.80), ciprofloxacin (OR 6.0; 95% CI 1.23–29.26), fluconazole (OR 4.59; 95% CI 1.55–13.52), and steroids (OR 4.13; 95% CI 1.11–15.39). Multivariate analysis showed that the determinants for fluconazole resistance were therapy with antituberculous drugs (OR 3.61; 95% CI 1.08–12.07;P=0.03) and one of the following: previous tuberculosis (OR 3.53; 95% CI 1.08–14.57;P=0.03) or fluconazole exposure (OR 3.41; 95% CI 1.10–10.54). Findings from this study indicate that treatment with antituberculous drugs, previous tuberculosis, and fluconazole exposure are the strongest determinants for development of oropharyngeal colonization or infection by fluconazole-resistant Candida strains in HIV-infected patients.     

Carriage of Antibiotic-Resistant Streptococcus pneumoniae in Greek Infants and Toddlers

The prevalence, resistance patterns and serotypes of antibiotic-resistant Streptococcus pneumoniae strains recovered from Greek carriers under 24 months of age were studied. From February 1997 to April 1998, nasopharyngeal cultures were performed in 1269 children (ages 2–23 months, median 11 months) living in various areas of central and southern Greece. Resistance (including both intermediate and resistant isolates) to one or more antimicrobial agents was found in 132 of the 421 (31%) Streptococcus pneumoniae isolates, as follows: penicillin, 9% intermediate, 7.6% resistant; cefotaxime, 5.2% intermediate, 0.5% resistant; erythromycin, 0.7% intermediate, 18.1% resistant; clindamycin, 0.2% intermediate, 12.4% resistant; tetracycline, 0.7% intermediate, 16.4% resistant; chloramphenicol, 12.4% resistant; and trimethoprim-sulfamethoxazole, 3.8% intermediate, 14.3% resistant. The MICs of penicillin for 66% of the penicillin-nonsusceptible pneumococci were 1–4 μg/ml. Multidrug resistance was found in 64% of penicillin-nonsusceptible and 37% of penicillin-susceptible strains. Sixty-two percent of the penicillin-susceptible, multidrug-resistant strains belonged to serotype 6B and were resistant to all five non-β-lactam agents tested. This notable serotype 6B resistance pattern was described for the first time in a previous study performed from December 1995 to February 1996 in the city of Patras, southwestern Greece. Seventy-two percent of antibiotic-resistant isolates belonged to serotypes 6B, 9V, 14, 18C, 19F and 23F. These results document the spread of resistant pneumococcal strains in central and southern Greece, many of which are multidrug resistant.