MICs Of Clindamycin Research Articles

P-1493. In Vitro Activity of Clindamycin in Comparison with Linezolid Versus Streptococcus pyogenes Clinical Isolates Recovered from Patients in Manitoba, Canada

Abstract Background Clindamycin is often used in combination with a beta-lactam antimicrobial for the treatment of serious infections caused by Streptococcus pyogenes (e.g., necrotizing fasciitis, streptococcal toxic shock syndrome) due to its ability to suppress bacterial toxin synthesis. Of concern, increasing clindamycin resistance among S. pyogenes isolates has been reported in many parts of the world. The purpose of this study was to compare the in vitro activity of clindamycin with linezolid versus a contemporary Canadian collection of S. pyogenes clinical isolates. Methods S. pyogenes isolates from wounds and sterile body sites (one per patient) were obtained from three microbiology laboratories in Manitoba, Canada between December, 2022 and November, 2023. Susceptibility testing was performed by broth microdilution as described by CLSI, with MICs interpreted using current CLSI breakpoints. A D-test was set up for each isolate to investigate whether inducible clindamycin resistance was present. Results Susceptibility testing was performed on 140 S. pyogenes clinical isolates. The isolates were obtained from wound swabs (n = 71, 50.7%), sterile fluid/tissue samples (n = 27, 19.3%), and blood cultures (n = 42, 30.0%). All isolates were fully susceptible in vitro to penicillin and vancomycin. The proportion of isolates testing susceptible to clindamycin and linezolid was 82.9% and 100%, respectively. All of the clindamycin-resistant isolates demonstrated an inducible resistance phenotype (clindamycin MIC ≤0.12 ug/mL in all cases). Clindamycin susceptibility was similar for isolates from wound swabs (81.7%) and isolates from blood/sterile sites (84.1%). Conclusion The proportion of S. pyogenes isolates testing susceptible to clindamycin was 82.9%. All clindamycin resistant isolates demonstrated an inducible resistance phenotype. Further data are required to determine whether inducible clindamycin resistance is clinically relevant when clindamycin is used as adjunctive therapy (in combination with a beta-lactam) for serious S. pyogenes infections. All isolates tested remained susceptible to linezolid. Disclosures All Authors: No reported disclosures

Pharmacokinetic interaction between rifampicin and clindamycin in staphylococcal osteoarticular infections

Oral combination of clindamycin and rifampicin is relevant for the treatment of staphylococcal osteoarticular infection (SOAIs). However, rifampicin induces CYP3A4, suggesting a pharmacokinetic interaction with clindamycin with unknown pharmacokinetic/pharmacodynamic (PK/PD) consequences. This study aimed to quantify clindamycin PK/PD markers before and during rifampicin co-administration in SOAI. Patients with SOAI were included. After initial intravenous antistaphylococcal treatment, oral therapy was started with clindamycin (600 or 750 mg t.i.d.), followed by addition of rifampicin 36 h later. Population PK analysis was performed using the SAEM algorithm. PK/PD markers were compared with and without rifampicin co-administration, each patient being his own control. In 19 patients, clindamycin median (range) trough concentrations were 2.7 (0.3-8.9) mg/L and <0.05 (<0.05-0.3) mg/L before and during rifampicin administration, respectively. Rifampicin co-administration increased clindamycin clearance by a factor 16 and reduced the AUC0-8h/MIC by a factor 15 (P < 0.005). Clindamycin plasma concentrations were simulated for 1000 individuals, without and with rifampicin. Against a susceptible Staphylococcus aureus strain (clindamycin MIC 0.0625 mg/L), >80% of individuals would reach all proposed PK/PD targets without co-administration of rifampicin, even with low clindamycin dose. For the same strain, when rifampicin was co-administered, the probability to reach clindamycin PK/PD targets dropped to 1% for %fT>MIC=100% and to 6% for AUC0-24h/MIC > 60, even with high clindamycin dose. Rifampicin co-administration with clindamycin has a high impact on clindamycin exposure and PK/PD targets in SOAI, which could result in clinical failure even for fully susceptible strains.

Synergistic antimicrobial activity of melittin with clindamycin on the expression of encoding exfoliative toxin in Staphylococcus aureus

Staphylococcus aureus is an opportunistic human pathogens, with the ability to produce a series of virulence factors that contribute to the severity of infections. Exfoliative toxins (ETs) are one of the important virulence factors that participating in staphylococcal scalded skin syndrome. Melittin has different biological activities, comprising of antiviral, broad spectrum antibacterial, antiprotozoal, antifungal and anti-inflammatory effects. Twelve clinical isolates of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) were obtained from wound infection in the burn patients. The MIC plus three sub-inhibitory concentrations (I, II and III) of clindamycin and melittin were tested. Next, the synergistic effects of melittin and clindamycin were evaluated using the broth microdilution checkerboard assay. The detection of exfoliative toxin A and B genes were examined by PCR method. Then the effects of sub-MIC melittin on the expression levels of eta and etb were assessed by quantitative real-time PCR (qRT-PCR) assay. Melittin MIC values against MRSA and MSSA planktonic cells were 0.25–0.5 and 0.25–1 μg/ml, respectively. The clindamycin MIC values against MRSA and MSSA were between 0.5 and 8 μg/ml and 0.5–2 μg/ml, respectively. The results of the time-kill kinetics assay (3.5log10 and 3log10) against MSSA and MRSA planktonic cells were determined within 24 h using melittin. The mean expression of eta in MRSA and MSSA was significantly downregulated to approximately 3.5 and 4 fold, respectively. Moreover, the mean expression of etb in MRSA and MSSA were significantly downregulated to approximately 2.5 and 3 fold, respectively. Hemolytic assay showed that the extracted melittin indicates a strong hemolytic activity (HD50 = 2 μg/ml). Melittin at 0.5 μg/ml induced cell lysis and stimulated the formation of vesicles in S. aureus strains. Melittin could reduce the expression of eta and etb as encoding exfoliative toxin A and B genes. This component appears to be a good candidate for the treatment of MRSA and MSSA strains. So, melittin in combination with clindamycin can be classified as a complementary treatment of wound infections in burn patients.

New genetic context of lnu(B) composed of two multi-resistance gene clusters in clinical Streptococcus agalactiae ST-19 strains

BackgroundClindamycin is a lincosamide antibiotic used to treat staphylococcal and streptococcal infections. Reports of clinical Streptococcus agalactiae isolates with the rare lincosamide resistance/macrolide susceptibility (LR/MS) phenotype are increasing worldwide. In this study, we characterised three clinical S. agalactiae strains with the unusual L phenotype from China.MethodsThree clinical S. agalactiae strains, Sag3, Sag27 and Sag4104, with the L phenotype were identified from 186 isolates collected from 2016 to 2018 in Shanghai, China. The MICs of clindamycin, erythromycin, tetracycline, levofloxacin, and penicillin were determined using Etest. PCR for the lnu(B) gene was conducted. Whole genome sequencing and sequence analysis were carried out to investigate the genetic context of lnu(B). Efforts to transfer lincomycin resistance by conjugation and to identify the circular form by inverse PCR were made.ResultsSag3, Sag27, and Sag4104 were susceptible to erythromycin (MIC ≤0.25 mg/L) but resistant to clindamycin (MIC ≥1 mg/L). lnu(B) was found to be responsible for the L phenotype. lnu(B) in Sag3 and Sag27 were chromosomally located in an aadE-spw-lsa(E)-lnu(B) resistance gene cluster adjacent to an upstream 7-kb tet(L)-cat resistance gene cluster. Two resistance gene clusters were flanked by the IS6-like element, IS1216. Sag4104 only contained partial genes of aadE-spw-lsa(E)-lnu(B) resistance gene cluster and was also flanked by IS1216.ConclusionThese results established the presence of the L phenotype associated with lnu(B) in clinical S. agalactiae isolates in China. The lnu(B)-containing multi-resistance gene cluster possibly acts as a composite transposon flanked by IS1216 and as a vehicle for the dissemination of multidrug resistance among S. agalactiae.

Distribution of Clostridium Difficile Ribotypes in Macedonian Patients and their Antimicrobial Susceptibility.

BACKGROUND:Clostridium difficile is a major nosocomial pathogen. In Europe, this bacterium is mostly characterised by PCR ribotyping. Most of the Clostridium difficile infections (CDI) are treated with vancomycin or metronidazole, although prolonged antibiotic use is considered as one of the main risk factors for CDI.AIM:This study aimed to detect the presence of various C. difficile ribotypes in hospitalised patients and to investigate their toxigenicity and antibiotic susceptibility.MATERIAL AND METHODS:All stool samples obtained from each patient were inoculated on Columbia blood agar and cycloserine cefoxitine fructose agar (CCFA) for isolation of C. difficile. Glutamate dehydrogenase and toxins A and B were investigated by immunochromatographic tests. Final confirmation of the isolates was performed by Vitek 2 and MALDI-TOF. A total of 21 isolates were collected for further investigation. PCR ribotyping was performed as described by Janezic and Rupnik. PCR ribotype profiles were analysed using software (Bionumerics, Applied Maths). Antibiotic susceptibility was determined by E-tests for metronidazole, vancomycin, tetracycline, clindamycin, erythromycin, imipenem, ciprofloxacin and moxifloxacin.RESULTS:About 48% of C. difficile isolates belonged to ribotype 001/072. So, this ribotype was the most common ribotype in this study. The remaining 52% of C. difficile isolates consisted of 10 different ribotypes: 017, SLO 160, SLO 187, SLO 120, 255/258, 014/020, 046, 002, 070 and 027. Furthermore, 20 (95.2 %) out of 21 isolates of C. difficile were toxigenic. Toxins A and B were detected simultaneously in 90.5 % of C. difficile isolates. Two isolates from the ribotype 017 were toxin B positive only. Treatments with any of the following antimicrobials: clindamycin, erythromycin, ciprofloxacin and moxifloxacin (as well as many other antibiotics), could be a risk factor for CDI due to the high resistance of the strains in this study. About 90% of the strains from the most common ribotype 001/072 have MICs for clindamycin and erythromycin > 256 µg/ml.CONCLUSION:All strains isolated are highly resistant to ciprofloxacin. All strains were susceptible to vancomycin (median MIC was 0.63 µg/ml) and metronidazole (median MIC was 0.084 µg/ml), so these two antimicrobials remain optimal treatment option for CDI.

The presence of antibiotic resistance genes and bft genes as well as antibiotic susceptibility testing of Bacteroides fragilis strains isolated from inpatients of the Infant Jesus Teaching Hospital, Warsaw during 2007–2012

The purpose of this study was to assess drug susceptibility of clinical B. fragilis strains and to determine any correlation between drug resistance and the presence of specific genes. Antimicrobial susceptibility was assessed using E-tests. All isolates were analyzed with the PCR technique for the presence of antibiotic resistance genes (cepA, cfxA, cfiA, ermF, ermB, ermG, nim), insertion sequences elements (IS1186, IS1187, IS1188, IS942), and enterotoxin-encoding genes (bft). Susceptibility tests yielded the following rates of resistance to the evaluated antibiotics: penicillin G (100%), clindamycin (22.5%), cefoxitin (6.3%), amoxicillin/clavulanic acid (1.8%). All strain were susceptible to imipenem, and metronidazole. The following antibiotic resistance genes were detected in the evaluated isolates: cepA (in 96.4% of isolates), cfxA (in 12.6%), cfiA (in 1.8%), and ermF (in 25.2%). Genes ermB, ermG, and nim were not found. The presence of the cepA gene showed no correlation with the penicillin G MIC. However, we observed a high correlation between cefoxitin MIC values and the presence of gene cfxA as well as a nearly complete correlation between clindamycin MIC values and the presence of gene ermF. The presence of a bft gene was detected in 14.4% of the analyzed B. fragilis isolates; with the bft-1 allele found in 75%, bft-2 in 25%, and bft-3 in none of the isolates. Antibiotic susceptibility profiles of enterotoxin gene-positive isolates in our study did not differ from those of enterotoxin gene-negative isolates.

Biocide tolerance, phenotypic and molecular response of lactic acid bacteria isolated from naturally-fermented Aloreña table to different physico-chemical stresses

Lactic acid bacteria (LAB) isolated throughout the fermentation process of Aloreña table olives were found to be resistant at least to three antibiotics (Casado Muñoz et al., 2014); however, most were sensitive to the biocides tested in this study (with minimum inhibitory concentrations [MIC] below the epidemiological cut-off values). 2–15% of the isolates were found to be biocide resistant: Leuconostoc Pseudomesenteroides, which were resistant to hexachlorophene, and Lactobacillus pentosus to cetrimide and hexadecylpiridinium.We analyzed the effect of different physico-chemical stresses, including antimicrobials, on the phenotypic and genotypic responses of LAB, providing new insights on how they become resistant in a changing environment. Results indicated that similar phenotypic responses were obtained under three stress conditions: antimicrobials, chemicals and UV light. Susceptibility patterns to antibiotics changed: increasing MICs for ampicillin, chloramphenicol, ciprofloxacin, teicoplanin and tetracycline, and decreasing the MICs for clindamycin, erythromycin, streptomycin and trimethoprim in most strains. Statistically, cross resistance between different antibiotics was detected in all stress conditions. However, expression profiles of selected genes involved in stress/resistance response (rpsL, recA, uvrB and srtA) differed depending on the stress parameter, LAB species and strain, and the target gene.We conclude that, despite the uniform phenotypic response to stresses, the repertoire of induced and repressed genes differs. So, a search for a target to improve stress tolerance of LAB, especially those of importance as starter/protective cultures or probiotics, may depend on the individual screening of each strain, even though we could predict the antibiotic phenotypic response to all stresses.

First report of macrolide resistance gene erm(T) harbored by a novel small plasmid from Erysipelothrix rhusiopathiae.

The macrolide resistance gene erm(T) was identified for the first time in a porcine Erysipelothrix rhusiopathiae isolate from swine in China. The novel 3,749-bp small plasmid pER29, which carries erm(T), had a G+C content of 31% and four distinct open reading frames. The presence of pER29 increased by at least 128-fold the MICs of clindamycin and erythromycin for E. rhusiopathiae. The fitness cost of pER29 could be responsible for the low frequency of erm(T) in E. rhusiopathiae.

Resistance behaviour of inducible clindamycin-resistant staphylococci from clinical samples and aquatic environments.

In this study, the species diversity of staphylococci with inducible resistance to macrolides, lincosamides and streptogramin B (MLSB) isolated from clinical samples, sewage and river water was investigated. Inducible clindamycin resistance was tested using a D-test and macrodilution assays. Inducible cross-resistance (iMLSB phenotype) was examined by PCR of erm gene classes A, B, C, F, G, Q, T and 43. Although ermC was the most frequently detected resistance gene in iMLSB phenotypes of environmental staphylococci (61.2%), resistance genes encoding iMLSB were more diverse than in staphylococci from hospital samples. In 22.4% of iMLSB staphylococci from aquatic environments, none of the eight tested erm genes was found. Those isolates and erm43-expressing Staphylococcus lentus displayed low erythromycin MICs (3-16 µg ml(-1)) compared with ermC-positive environmental staphylococci (≥256 µg ml(-1)). In contrast to clinical isolates with clearly defined resistance behaviour, resistance patterns against MLSB and MICs for clindamycin of environmental isolates were more diverse. Although the abundance of iMLSB staphylococci in the aquatic environment was lower than in staphylococci from hospital samples, the diversity of resistance genes encoding this phenotype seemed to be higher. Oleandomycin is the best marker to correlate iMLSB phenotype and the respective erm gene. The phenotypical behaviour of environmental isolates may differ from the resistance pattern of clinical iMLSB staphylococci expressing ermA or ermC, and this should be considered for successful treatment of infections.

MRSA Pediatric clone expressing ermC plus lnuA genes causing nosocomial transmission and healthcare workers colonization in a neonatal intensive care unit

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of both nosocomial and community-acquired infections. We describe an outbreak caused by the MRSA Pediatric clone expressing an unusual lincosamide resistant phenotype. Between January and May 2006, an MRSA outbreak was detected at the Neonatal Unit of Hospital Interzonal General de Agudos “Evita”, Buenos Aires Province, Argentina that affected ten patients. Seven isolates from seven patients plus five MRSA recovered from health care workers (nasal carriage) were studied. Two phenotypes were observed: (i) ELCi (10), resistance to erythromycin and lincomycin and inducible resistance to clindamycin; (ii) ELiCi (2), resistance to erythromycin and inducible resistance to lincomycin and clindamycin. All 12 MRSA were resistant to oxacillin, erythromycin and gentamicin. Isolates expressing the ELCi-phenotype showed lincomycin MIC values between 16 and 32mg/L, while the remaining 2 isolates with ELiCi-phenotype presented a MIC value of 0.5mg/L. No differences were observed between the clindamycin MIC values in both phenotypes, ranging 0.25–0.5mg/L. Isolates showing ELCi-phenotype harbored ermC plus lnuA genes, and the other two only ermC gene. All 12 isolates were genetically related and belonged to the Pediatric clone (ST100) harboring a new variant of SCCmecIV. This is the first MRSA outbreak expressing an unusual ELCi phenotype due to a combination of ermC plus lnuA genes.

Answer to March 2014 Photo Quiz

Answer: Bacteremia caused by Tsukamurella tyrosinosolvens. The isolate was definitively identified as Tsukamurella tyrosinosolvens via DNA sequence analysis of the 16S rRNA gene. Similar to those of aerobic actinomycetes such as Gordonia spp., Rhodococcus spp., and Nocardia spp., cell walls of Tsukamurella spp. contain mycolic acids and allow for a weakly positive reaction upon modified acid-fast staining (1). The longer bacillary form of Tsukamurella spp. potentiates preliminary differentiation from Gordonia spp. (short coryneform bacilli) and Rhodococcus spp. (some coccoid forms) (1). Additional phenotypic delineation of Tsukamurella spp. can be guided with demonstration of growth in lysozyme (also Nocardia spp.) and specialized characterization of mycolic acid and menaquinone chemistry. However, molecular techniques, particularly those centered on gene sequencing, are necessary for definitive identification of aerobic actinomycetes to both the genus and species levels (1). Tsukamurella spp. have been documented to be etiologies of clinical disease, with reports increasing in recent years. Within a 12-year period at a university hospital, Liu et al. (2) rank ordered isolates of T. tyrosinosolvens, Tsukamurella spumae, and Tsukamurella pulmonis in the context of keratitis or catheter-related bloodstream infection. On rare occasions, Tsukamurella paurometabola has also been implicated in clinical disease (1). Specific to T. tyrosinosolvens, a recent report by Karunakaran et al. (3) typified a common clinical presentation. A catheter-related bloodstream infection in a 51-year-old hematology patient was resolved via catheter removal and an imipenem regimen. T. tyrosinosolvens has also been implicated in cerebellar abscess (4) and presentations of respiratory disease (5, 6). The two preceding citations shared an underlying status of immunosuppression, ranging from antecedent lung transplantation to delivery of chemotherapeutic agents by a central venous catheter. The cerebellar abscess presented in an immunocompetent patient with predisposing chronic otitis media that was refractory to penicillin and cephalosporin agents. Antimicrobial susceptibility testing was not performed on this isolate, due to the inconclusive utility of the Clinical and Laboratory Standards Institute standards for testing aerobic actinomycetes other than Nocardia spp. (7). Liu et al. (2), in a limited data set, suggested that T. pulmonis yielded higher MICs for clindamycin, erythromycin, vancomycin, and tetracycline than other Tsukamurella spp. Antimicrobials with lower MICs across multiple species included gentamicin, levofloxacin, and ciprofloxacin. Similar to data reported by Karunakaran et al. (3), our patient defervesced quickly upon removal of the peripherally inserted central catheter (PICC) line and initiation of antimicrobial therapy. The patient was discharged to home on parenteral vancomycin therapy. (See page 711 in this issue [doi:10.1128/JCM.03462-12] for photo quiz case presentation.)

Can interchangeability of lincosamides be assumed in clinical practice? Comparative MICs of clindamycin and lincomycin for Streptococcus pyogenes, Streptococcus agalactiae and Staphylococcus aureus

Can interchangeability of lincosamides be assumed in clinical practice? Comparative MICs of clindamycin and lincomycin for Streptococcus pyogenes, Streptococcus agalactiae and Staphylococcus aureus

84 Azithromycin resistance in Prevotella species isolated from CF patients

Objective: The use of chronic azithromycin treatment has been linked with increased macrolide resistance. Although, Prevotella spp. are one of the most common anaerobes detected in CF pulmonary samples, the effect of chronic azithromycin exposure on this genus is unknown. The aims of this study were to i. investigate if resistance is associated with azithromycin prescription ii. compare azithromycin and clindamycin MICs between Prevotella isolates cultured from CF patients and healthy control subjects. Methods: Isolates were grouped according to source and patient prescription of azithromycin: CF (currently prescribed), n = 27; CF (not currently prescribed), n = 30; healthy controls (none), n = 17. Susceptibility was determined by Etest® and MICs compared between groups using the Mann-Whitney test. Results: CF isolates had significantly higher azithromycin and clindamycin MICs compared to the healthy control isolates (P < 0.001). Current prescription of azithromycin was associated with significantly higher azithromycin MICs (P= 0.016) in the CF isolates. CF isolates (not currently prescribed) had significantly higher azithromycin MICs (P= 0.009) compared to isolates from healthy control subjects. Isolates from the 2 CF groups had equal resistance to clindamycin (P= 0.228). Conclusions: CF patients harbour isolates with increased resistance to azithromycin and clindamycin. Azithromycin resistance is associated with current azithromycin prescription. Work supported by a Department of Employment and Learning, NI (DEL) studentship to L. Sherrard and by HSC Research and Development, Public Health Agency, NI and the Medical Research Council through a US-Ireland Partnership Grant.

85 Investigation into the antibiotic resistome of the Streptococcus genus from adult cystic fibrosis patients

Objective: The use of chronic azithromycin treatment has been linked with increased macrolide resistance. Although, Prevotella spp. are one of the most common anaerobes detected in CF pulmonary samples, the effect of chronic azithromycin exposure on this genus is unknown. The aims of this study were to i. investigate if resistance is associated with azithromycin prescription ii. compare azithromycin and clindamycin MICs between Prevotella isolates cultured from CF patients and healthy control subjects. Methods: Isolates were grouped according to source and patient prescription of azithromycin: CF (currently prescribed), n = 27; CF (not currently prescribed), n = 30; healthy controls (none), n = 17. Susceptibility was determined by Etest® and MICs compared between groups using the Mann-Whitney test. Results: CF isolates had significantly higher azithromycin and clindamycin MICs compared to the healthy control isolates (P < 0.001). Current prescription of azithromycin was associated with significantly higher azithromycin MICs (P= 0.016) in the CF isolates. CF isolates (not currently prescribed) had significantly higher azithromycin MICs (P= 0.009) compared to isolates from healthy control subjects. Isolates from the 2 CF groups had equal resistance to clindamycin (P= 0.228). Conclusions: CF patients harbour isolates with increased resistance to azithromycin and clindamycin. Azithromycin resistance is associated with current azithromycin prescription. Work supported by a Department of Employment and Learning, NI (DEL) studentship to L. Sherrard and by HSC Research and Development, Public Health Agency, NI and the Medical Research Council through a US-Ireland Partnership Grant.

Scarce detection of mobile erm genes associated with tetQ in Bacteroides and Parabacteroides from Costa Rica

The frequency of finding of clindamycin-resistant anaerobic bacteria in clinical samples has doubled from 2008 to 2010 in Costa Rica. To determine whether this increase is due to dissemination of erm genes aided by tetQ elements, we analyzed 100 isolates of Bacteroides or Parabacteroides from a regional hospital, a national hospital, and the community. Antimicrobial susceptibilities were recorded with a broth micro-dilution method and erm genes were detected by PCR and Southern blotting. In addition, plasmid isolation and mating experiments were performed to clarify the location and mobility of the detected erm genes. Resistance to clindamycin was by far more frequent in the regional hospital (72%) than in the national hospital (29%) and the community (26%). Resistance to tetracycline was even more common, with the community (85%) outweighing the hospitals (71–72%). While MIC of clindamycin were higher in the hospitals than in the community (P < 0.05), the opposite was seen for tetracycline (P < 0.0001). Of the sought-after genes, only ermG (n = 2), ermA (n = 1), and ermF (n = 1) were detected in the hospitals and ermF in the community (n = 2). In opposition to the low frequency of finding of erm genes, 71% of the isolates were positive for tetQ. None of the detected genes were encoded on plasmids. Only three isolates from the hospitals transferred their erm genes laterally. By contrast, 13 hospital isolates and two community isolates transferred tetQ. Despite the widespread finding of tetracycline-resistant tetQ-positive bacteria, mobile erm genes were rare in our bacterial collection. We conclude that the detected erm genes are likely not included in typical conjugative transposons of Bacteroides and Parabacteroides.

Macrolide and tetracycline resistance in clinical strains of Streptococcus agalactiae isolated in Tunisia

Between 2007 and 2009, 226 clinical strains of Streptococcus agalactiae, recovered from female genital specimens and from gastric fluid or ear specimens from infected newborns, were isolated at the Laboratory of Microbiology of Charles Nicolle Hospital of Tunis. They were investigated to determine the prevalence of antibiotic resistance and to characterize the mechanisms of resistance to macrolide and tetracycline. All strains were susceptible to penicillin, ampicillin and quinupristin-dalfopristin. They were resistant to chloramphenicol (3.1%), rifampicin (19.1%), erythromycin (40%) and tetracycline (97.3%); 3.1% were highly resistant to streptomycin and 1.3% to gentamicin. Among the erythromycin-resistant isolates, 78.7% showed a constitutive macrolide-lincosamide-streptogramin B (MLS(B)) phenotype with high-level resistance to macrolides and clindamycin (MIC(50) >256 µg ml(-1)), 10% showed an inducible MLS(B) phenotype with high MICs of macrolides (MIC(50) >256 µg ml(-1)) and low MICs of clindamycin (MIC(50)=8 µg ml(-1)) and 2.2% showed an M phenotype with a low erythromycin-resistance level (MIC range=12-32 µg ml(-1)) and low MICs of clindamycin (MIC range: 0.75-1 µg ml(-1)). All strains were susceptible to quinupristin-dalfopristin and linezolid (MIC(90): 0.75 µg ml(-1) for each). MLS(B) phenotypes were genotypically confirmed by the presence of the erm(B) gene and the M phenotype by the mef(A) gene. Resistance to tetracycline was mainly due to the tet(M) gene (93.1%) encoding a ribosome protection mechanism. This determinant is commonly associated with the conjugative transposon Tn916 (P≤0.0002). tet(O) and tet(T) existed in a minority (2.2% and 0.4%, respectively). The efflux mechanism presented by tet(L) was less frequently present (4.5%). No significant association was found between erm(B) and tet(M) genes.

Unusual resistance patterns in macrolide-resistant Streptococcus pyogenes harbouring erm(A)

We identified erm(A)-harbouring Streptococcus pyogenes that expressed three variant phenotypes: (1) low-level resistance to erythromycin (MICs 1-4 mg/L) but high azithromycin MICs in absolute terms (16-64 mg/L; n=6); (2) same as (1) but with a high clindamycin MIC (256 mg/L; n=1); and (3) high-level constitutive MLS (cMLS) resistance (n=1). Here we analysed the genetic basis of these novel phenotypes. The presence of erm(A) and the absence of macrolide/lincosamide resistance genes erm(B), mef and cfr were confirmed by PCR. erm(A), 23S rRNA, L4 and L22 genes were sequenced. Mutant erm(A) genes were cloned and electrotransformed into the macrolide-susceptible Escherichia coli AG100A. Clonality was determined by emm typing and PFGE. Effects of the identified mutations on free energy changes (DeltaG) and putative configurations of the leader sequence were studied in silico. Point mutations (G98A, A137C, C140T and G205A) were observed in the erm(A) regulatory region of all eight erm(A)-harbouring S. pyogenes. Five and two isolates belonged to emm77 and emm89 clones, respectively, and one isolate was an emm1. E. coli transformed with mutant erm(A) harbouring G98A, A137C or C140T mutations (phenotypes 1 and 2) did not express high-level azithromycin or clindamycin resistance. However, cMLS resistance was clearly observed in transformants with erm(A) harbouring both A137C and G205A mutations (phenotype 3). In silico analysis showed that DeltaG was minor except for the G205A mutation. Secondary structure predictions further showed that the A137C and G205A mutations together abolished the hairpin sequestering the ribosome-binding and initiation sites of the erm(A) gene, explaining the cMLS phenotype 3. We report point mutations in the erm(A) regulatory region leading to constitutive methylase expression and the presence of additional, as yet unidentified mechanisms mediating high-level azithromycin and clindamycin resistance in erm(A)-harbouring S. pyogenes.

Trans‐Cinnamaldehyde from Cinnamomum zeylanicum Bark Essential Oil Reduces the Clindamycin Resistance of Clostridium difficile in vitro

Therapy with antimicrobial drugs, such as clindamycin, that perturb the intestinal flora but fail to inhibit growth of other microorganisms can permit the proliferation of Clostridium difficile and the elaboration of exotoxin. Therefore, there has been increasing interest in the use of inhibitors of antibiotic resistance for use in combination therapy. The essential oil of Cinnamomum zeylanicum bark enhanced the bactericidal activity of clindamycin and decreased the minimum inhibitory concentration of clindamycin required for a toxicogenic strain of C. difficile. Thin-layer chromatography (TLC) analysis of the essential oil separated a fraction (R(f) = 0.54) that was the most effective at enhancing the clindamycin antimicrobial activity. Using gas liquid chromatography and known standards, the active fraction was identified as trans-cinnamaldehyde (3-phenyl-2-Propenal). Combinations of clindamycin and trans-cinnamaldehyde were tested to determine the fractional inhibitory concentration (FIC) index by conventional checkerboard titration. The FIC index for C. difficile was found to be 0.312, which confirmed the synergistic actions of clindamycin and trans-cinnamaldehyde. The presence of 20 microg/mL of trans-cinnamaldehyde decreased the MIC of clindamycin for C. difficile 16-fold, from 4.0 to 0.25 microg/mL. These results signify that low concentrations of trans-cinnamaldehyde elevate the antimicrobial action of clindamycin, suggesting a possible clinical benefit for utilizing these natural products for combination therapy against C. difficile.

Expression of the mef (E) Gene Encoding the Macrolide Efflux Pump Protein Increases in Streptococcus pneumoniae with Increasing Resistance to Macrolides

Active macrolide efflux is a major mechanism of macrolide resistance in Streptococcus pneumoniae in many parts of the world, especially North America. In Canada, this active macrolide efflux in S. pneumoniae is predominantly due to acquisition of the mef(E) gene. In the present study, we assessed the mef(E) gene sequence as well as mef(E) expression in variety of low- and high-level macrolide-resistant, clindamycin-susceptible (M-phenotype) S. pneumoniae isolates (erythromycin MICs, 1 to 32 microg/ml; clindamycin MICs, < or = 0.25 microg/ml). Southern blot hybridization with mef(E) probe and EcoRI digestion and relative real-time reverse transcription-PCR were performed to study the mef(E) gene copy number and expression. Induction of mef(E) expression was analyzed by Etest susceptibility testing pre- and postincubation with subinhibitory concentrations of erythromycin, clarithromycin, azithromycin, telithromycin, and clindamycin. The macrolide efflux gene, mef(E), was shown to be a single-copy gene in all 23 clinical S. pneumoniae isolates tested, and expression post-macrolide induction increased 4-, 6-, 20-, and 200-fold in isolates with increasing macrolide resistance (erythromycin MICs 2, 4, 8, and 32 microg/ml, respectively). Sequencing analysis of the macrolide efflux genetic assembly (mega) revealed that mef(E) had a 16-bp deletion 153 bp upstream of the putative start codon in all 23 isolates. A 119-bp intergenic region between mef(E) and mel was sequenced, and a 99-bp deletion was found in 11 of the 23 M-phenotype S. pneumoniae isolates compared to the published mega sequence. However, the mef(E) gene was fully conserved among both high- and low-level macrolide-resistant isolates. In conclusion, increased expression of mef(E) is associated with higher levels of macrolide resistance in macrolide-resistant S. pneumoniae.

Differences in the DNA sequence of the translational attenuator of several constitutively expressed erm(A) genes from clinical isolates of Streptococcus agalactiae

To study the regulatory region of a constitutively expressed erm(A) gene in Streptococcus agalactiae clinical isolates. Thirty clinical isolates of S. agalactiae which were cross-resistant to erythromycin and clindamycin and with a clindamycin MIC higher than that of erythromycin were studied by PCR, sequencing and molecular typing. PCR analysis revealed that all the strains harboured the erm(A) gene, either alone (26 isolates) or in combination with erm(B) (four isolates). Sequencing of the region upstream of erm(A) showed that all isolates possessed two types of genetic alteration. Most of the strains showed point mutations in the second leader peptide (mainly A137C) and, in four isolates (two clones), an insertion fragment with high homology to IS1381 and transposase genes was detected. Epidemiological analysis of strains indicated several clonal origins of isolates. The mutations described here are thought to result in increased or constitutive expression of the erm(A) gene.