Constitutive Resistance Phenotype Research Articles

CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS ISOLATED FROM BURN WOUND; STRONG ANTIBACTERIAL ACTIVITY OF PHAGE COCKTAIL AGAINST VANCOMYCIN INTERMEDIATE-RESISTANT STAPHYLOCOCCUS AUREUS

A universal problem is about spread of Staphylococcus aureus infections in burn patients. The present study aimed to learn about the molecular characteristics and the resistance pattern of S. aureus strains isolated from burn patients. In this cross-sectional study, we investigated 100 unique S. aureus isolated from burn patients by antimicrobial activity and biofilm formation and evaluated the effect of Complex Pyobacteriophage, a commercial bacteriophage cocktail, against the isolates mentioned above. Methicillin-resistant Staphylococcus aureus (MRSA) comprised 76%, and methicillin-susceptible Staphylococcus aureus (MSSA) comprised 24% of 100 S. aureus strains. The resistance rate among MRSA isolates was higher than compared of MSSA. Mupirocin resistance was found in 30% of isolates, with 28 (93.3%) and 2 (6.7%) strains of MRSA and MSSA, respectively, found. Vancomycin intermediate resistance in S. aureus (VISA) was 13% of MRSA strains. Two isolates were confirmed as vancomycin-resistant S. aureus (VRSA) strains and carried vanA. 31 and 62% of the total isolates showed inducible and constitutive resistance phenotypes. The rate of inducible and constitutive clindamycin resistance among MRSA strains was higher than MSSA strains. Biofilm production was detected in 66% of isolates. Strong, moderate, and weak producers accounted for 25%, 17%, and 24% of isolates. Phage analysis showed that 81% were susceptible to the phage cocktail, and only 19% were resistant to the phage cocktail. Our data indicated that VISA strains prevalence in the burn unit was mainly from S. aureus infections. Present work recommended that vancomycin treatment be closely monitored to prevent the spread of VISA and VRSA strains. Observations also highlighted the role of bacteriophage cocktails in eradicating S. aureus-related infections.

Antibiotics Susceptibility Profile of <i>Staphylococcus aureus</i> Clinical Isolates Collected in Hospitals in the City of Douala (Cameroon)

Introduction: Staphylococcus aureus is one of the most important agents involved in community and hospital-acquired infections. Due to the multi-drug resistance of strains to antibiotics, treatment to eradicate it is becoming more difficult and poses a global public health problem. Methodology: This was a cross-sectional study conducted from March to August 2020 in hospitals in the city of Douala, including all S. aureus isolates from diagnostic samples. Strain identification and antibiotic susceptibility testing were performed using the Vitek2 CompactTM (BioMerieux). Results: During the study period, 136 non-repetitive S. aureus strains were identified with a high frequency of methicillin-resistant S. aureus of 78.7%. The majority of the strains originated from the Douala General Hospital (66.9%) and was most frequently isolated from blood culture samples (55.1%). The study of biochemical characteristics showed that most of the strains identified had between 87% and 99% homology with the reference strain. The most active antibiotics were Quinupristin/Dalfopristin (94.2%), Linezolid (87.8%) and Vancomycin (84.2%). Methicillin resistance was associated with decreased susceptibility of S. aureus to other antibiotics such as Gentamycin (44.9%), Erythromycin (38.2%), Tetracycline (38.3%), Trimethoprim (21.4%), Ciprofloxacin (19.1%) and Levofloxacin (24.0%). Inducible MLSb and constitutive resistance phenotypes were identified with 26.7% and 22.8% respectively. Conclusion: The sensitivity of S. aureus strains differs from one antibiotic family to another, and remains good for molecules that are not available in our context. The high frequency of Methicillin-Resistant S. aureus shows the continuous progression of multi-resistant strains of S. aureus and their decreased sensitivity to usual antibiotics becomes more and more alarming.

The Mediator kinase module serves as a positive regulator of salicylic acid accumulation and systemic acquired resistance.

In plants, the calmodulin-binding transcription activators (CAMTAs) are required for transcriptional regulation of abiotic and biotic stress responses. Among them, CAMTA3 in Arabidopsis has been intensively studied and shown to function redundantly with CAMTA1 and CAMTA2 to negatively regulate plant immunity. The camta1/2/3 triple mutant accordingly exhibits severe dwarfism due to autoimmunity. Here, through a suppressor screen using camta1/2/3 triple mutant, we found that a mutation in Cyclin-Dependent Kinase 8 (CDK8) partially suppresses the dwarfism and constitutive resistance phenotypes of camta1/2/3. CDK8 positively regulates steady-state salicylic acid (SA) levels and systemic required resistance (SAR). The expression of SA biosynthesis genes such as ICS1 and EDS5 is down-regulated in cdk8 mutants under uninfected conditions, suggesting that CDK8 contributes to the transcriptional regulation of these SA pathway genes. Knocking out another Mediator kinase module member MED12 yielded similar defects including decreased steady-state SA level and compromised SAR, suggesting that the whole Mediator kinase module contributes to the transcriptional regulation of SA levels and SAR.

Microbiology quality, detection of enterotoxin genes and antimicrobial resistance of Staphylococcus aureus isolated from milk and Coalho cheese

This study evaluated the microbiological quality of milk and Coalho cheese, the prevalence of enterotoxin genes, antimicrobial resistance and determined an inducible MLSB resistance phenotype by the D-test in strains of Staphylococcus aureus isolated from these products. Seventy samples of milk and Coalho cheese were analyzed. S. aureus strains were identified by biochemical tests. The presence of se genes (sea-see) was tested by polymerase chain reaction. The antimicrobial sensitivity of S. aureus strains was evaluated for 13 antimicrobial drugs using the disk diffusion technique and the double-disk diffusion test (D-test) was performed to determine inducible resistance to lincosamide phenotype. The amount of toxin sufficient to cause foodborne diseases is generally observed when Staphylococcus populations exceed 105 CFU mL-1 g-1. In this study, none of the milk samples analyzed showed these counts; however, 73.3% (22/30) of Coalho cheese samples exceeded this value. A total of 109 isolates were identified as S. aureus. The presence of enterotoxin genes was detected in 25.7% of these isolates and amplified only for the sec gene. Most of the isolates (78.5%) were resistant to one or more antimicrobial agents. The D test showed that 25.0% of erythromycin-resistant isolates had the constitutive resistance phenotype, and 3.8% had the inducible resistance phenotype to clindamycin. These results indicate that these dairy products represent a health risk since these bacteria can cause foodborne diseases or may be a possible route for the transfer of antimicrobial resistance to humans.

Inducible clindamycin resistance among the clinical isolates of Staphylococcus aureus in a tertiary care hospital

Introduction: Clindamycin is commonly used to treat skin and soft tissue infections caused by Staphylococcus aureus particularly in methicillin-resistant S. aureus (MRSA) infections. Inducible clindamycin resistance (inducible macrolide–lincosamide–streptogramin B resistance [MLSB]) is a critical factor in antimicrobial susceptibility testing. This study was aimed to detect MLSB phenotypes and inducible clindamycin resistant phenotypes among S. aureus. Materials and Methods: A total of 649 S. aureus isolates from different clinical samples were evaluated, and methicillin-resistance was determined using the cefoxitin (30 μg) disc. Inducible resistance to clindamycin was detected by D-zone test as per Clinical and Laboratory Standards Institute guidelines. Results: Of the 649 isolates, 404 (62.2%) were methicillin-sensitive S. aureus (MSSA) and 245 (37.8%) were MRSA. Ninety-six (14.8%) isolates showed inducible clindamycin resistance, 175 (27%) showed constitutive resistance, and 55 (8.5%) isolates showed MS phenotype. Inducible and constitutive resistance phenotypes were found to be higher in MRSA (25% and 64.8%) as compared to MSSA (8.7% and 4.7%), respectively. Conclusions: Considering the higher prevalence of clindamycin resistance in MRSA isolates as compared MSSA isolates, routine D-test of MRSA isolates is strongly recommended to prevent treatment failure. Therefore, inducible clindamycin detection should be the part of S. aureus sensitivity testing in all the microbiology laboratories.

A study of inducible clindamycin resistance in erythromycin resistant clinical isolates of Staphylococcus species

Introduction: Erythromycin resistant Staphylococcus isolates with inducible resistance appear sensitive to clindamycin in in?vitro sensitivity testing. If clindamycin is used for treatment of such isolates, selection for constitutive mutants may lead to clinical failure. Current study was conducted to detect the presence of inducible clindamycin resistance in erythromycin resistant Staphylococcus isolates by disk diffusion method (D test). To correlate clindamycin resistance phenotypes with minimum inhibitory concentrations (MICs) of clindamycin, erythromycin, oxacillin and vancomycin among the isolates. To correlate various resistance phenotypes with methicillin resistance. Material and Methods: 150 non duplicate isolates of Staphylococcus species were identified and antibiotic susceptibility testing was done using Kirby Bauer’s disc diffusion method. MICs were determined using E?test for oxacillin, vancomycin, clindamycin and erythromycin using E?test strips (Himedia) Results: Among 150 staphylococcus clinical isolates, 96 were of S. aureus and 54 were coagulase negative Staphylococci (CONS). About 81.2% of the S.aureus isolates and 72.2% of the CONS were found to be methicillin resistant. Inducible clindamycin resistance was found in 39.3% of the isolates, constitutive resistance phenotype in 48% while 12.7% demonstrated MS phenotype. 18% and 11.3% of all the isolates had MICs for clindamycin between 0.01?0.06 μg/ml and 0.06?0.1 respectively. 12.5% had MIC ranging from 4?8 μg/ml while 58% had MIC > 8 μg/ml. Constitutive resistant phenotype (cMLS) was the predominant phenotype in methicillin resistant isolates. MS phenotype was the predominant among MSSA (methicillin sensitive S. aureus) while MSCNS (methicillin sensitive CONS) cMLS (46.7%) predominated. MIC of all erythromycin resistant isolates were ≥ 240 μg/ml. Nearly 16.7% of the cMLS and 57.9% of MS isolates were found to be oxacillin sensitive and 83% of iMLS and 83.3% of MS phenotype isolates were oxacillin resistant on MIC testing. 47.2% of cMLS and 73.6% of MS isolates had MIC ≤ 2 μg/ml for vancomycin and 52.7% of cMLS and 26.3% of MS isolates had MICs in intermediate range for vancomycin. Conclusions: D?testing might help clinicians to decide whether to use clindamycin in Staphylococcal infections when erythromycin resistance is present. Determination of MICs help to identify exact sensitivity profile of isolates in cases where clinical failure occurs due to misleading disk diffusion tests.DOI: http://dx.doi.org/10.3126/ajms.v6i6.11811 Asian Journal of Medical Sciences Vol.6(6) 2015 48-52

Evaluation of constitutive and inducible resistance to clindamycin in clinical samples of Staphylococcus aureus from a tertiary hospital.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have become common in hospitals and the community environment, and this wide resistance has limited patient treatment. Clindamycin (CL) represents an important alternative therapy for infections caused by S. aureus. Antimicrobial susceptibility testing using standard methods may not detect inducible CL resistance. This study was performed to detect the phenotypes of resistance to macrolides-lincosamides-streptogramin B (MLSB) antibiotics, including CL, in clinical samples of S. aureus from patients at a tertiary hospital in Santa Maria, State of Rio Grande do Sul, Brazil. One hundred and forty clinical isolates were submitted to the disk diffusion induction test (D-test) with an erythromycin (ER) disk positioned at a distance of 20mm from a CL disk. The results were interpreted according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). In this study, 29 (20.7%) of the 140 S. aureus samples were resistant to methicillin (MRSA), and 111 (79.3%) were susceptible to methicillin (MSSA). The constitutive resistance phenotype (cMLSB) was observed in 20 (14.3%) MRSA samples and in 5 (3.6%) MSSA samples, whereas the inducible resistance phenotype (iMLSB) was observed in 3 (2.1%) MRSA samples and in 8 (5.8%) MSSA samples. The D-test is essential for detecting the iMLSB phenotype because the early identification of this phenotype allows clinicians to choose an appropriate treatment for patients. Furthermore, this test is simple, easy to perform and inexpensive.

Inactivation of the Lipopeptide Antibiotic Daptomycin by Hydrolytic Mechanisms

The lipopeptide daptomycin is a member of the newest FDA-approved antimicrobial class, exhibiting potency against a broad range of Gram-positive pathogens with only rare incidences of clinical resistance. Environmental bacteria harbor an abundance of resistance determinants orthologous to those in pathogens and thus may serve as an early-warning system for future clinical emergence. A collection of morphologically diverse environmental actinomycetes demonstrating unprecedented frequencies of daptomycin resistance and high levels of resistance by antibiotic inactivation was characterized to elucidate modes of drug inactivation. In vivo studies revealed that hydrolysis plays a key role, resulting in one or both of the following structural modifications: ring hydrolysis resulting in linearization (in 44% of inactivating isolates) or deacylation of the lipid tail (29%). Characterization of the mechanism in actinomycete WAC4713 (a Streptomyces sp. with an MIC of 512 μg/ml) demonstrated a constitutive resistance phenotype and established daptomycin's circularizing ester linkage to be the site of hydrolysis. Characterization of the hydrolase responsible revealed it to be likely a serine protease. These studies suggested that daptomycin is susceptible to general proteolytic hydrolysis, which was further supported by studies using proteases of diverse origin. These findings represent the first comprehensive characterization of daptomycin inactivation in any bacterial class and may not only presage a future mechanism of clinical resistance but also suggest strategies for the development of new lipopeptides.

The Arabidopsis Resistance-Like Gene SNC1 Is Activated by Mutations in SRFR1 and Contributes to Resistance to the Bacterial Effector AvrRps4

The SUPPRESSOR OF rps4-RLD1 (SRFR1) gene was identified based on enhanced AvrRps4-triggered resistance in the naturally susceptible Arabidopsis accession RLD. No other phenotypic effects were recorded, and the extent of SRFR1 involvement in regulating effector-triggered immunity was unknown. Here we show that mutations in SRFR1 in the accession Columbia-0 (Col-0) lead to severe stunting and constitutive expression of the defense gene PR1. These phenotypes were temperature-dependent. A cross between srfr1-1 (RLD background) and srfr1-4 (Col-0) showed that stunting was caused by a recessive locus in Col-0. Mapping and targeted crosses identified the Col-0-specific resistance gene SNC1 as the locus that causes stunting. SRFR1 was proposed to function as a transcriptional repressor, and SNC1 is indeed overexpressed in srfr1-4. Interestingly, co-regulated genes in the SNC1 cluster are also upregulated in the srfr1-4 snc1-11 double mutant, indicating that the overexpression of SNC1 is not a secondary effect of constitutive defense activation. In addition, a Col-0 RPS4 mutant showed full susceptibility to bacteria expressing avrRps4 at 24°C but not at 22°C, while RLD susceptibility was not temperature-dependent. The rps4-2 snc1-11 double mutant showed increased, but not full, susceptibility at 22°C, indicating that additional cross-talk between resistance pathways may exist. Intriguingly, when transiently expressed in Nicotiana benthamiana, SRFR1, RPS4 and SNC1 are in a common protein complex in a cytoplasmic microsomal compartment. Our results highlight SRFR1 as a convergence point in at least a subset of TIR-NBS-LRR protein-mediated immunity in Arabidopsis. Based on the cross-talk evident from our results, they also suggest that reports of constitutive resistance phenotypes in Col-0 need to consider the possible involvement of SNC1.

Isolation of different erythromycin-resistance Streptococcus pneumoniae phenotypes in Piemonte Region

The frequency of erythromycin resistance in Streptococcus pneumoniae isolates from human specimens, in different Piemonte Region hospitals between 2005-2007, has been studied. Erythromycin-susceptible isolates were 137/198 and erythromycin-resistant 61/198.Among resistant S. pneumoniae isolates 26/61 belonged to constitutive resistance phenotype, 26/61 to M phenotype and 9/61 to inducible resistance phenotype.

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)

Distribuzione dei fenotipi di eritromicino-resistenza di Streptococcus pyogenes isolati in un’area circoscritta del Piemonte.

The frequency of erythromycin resistance in Streptococcus pyogenes isolated in different Piemonte Region hospitals between January and August 2006 has been studied. The incidence of erythromycin-resistance was 20.2%. Among erythromycin resistant S. pyogenes 13.8% belonged to constitutive resistance phenotype, 5.3% to M phenotype and 1.1% to inducible resistance phenotype.

In vitro Activity of Moxifloxacin Compared to Other Fluoroquinolones against Different Erythromycin-Resistant Phenotypes of Group A β-hemolytic Streptococcus

The aim of the present study was to evaluate the in vitro activity of moxifloxacin (BAY 12–8039) compared to ciprofloxacin, ofloxacin and sparfloxacin against 156 group A β-hemolytic Streptococcus strains. Sixty strains were macrolide-, lincosamide- and streptogramin-B-susceptible; 16 strains exhibited a constitutive resistance phenotype; 32 strains exhibited an inducible phenotype, and 48 strains were characterized by the M-type efflux-dependent phenotype. The minimum inhibition concentrations were determined by an agar dilution method according to NCCLS-approved guidelines. Moxifloxacin showed an enhanced activity compared with the other fluoroquinolones against the different macrolide-resistant phenotypes.

Erythromycin Resistance Genes in Group A Streptococci in Finland

Streptococcus pyogenes isolates (group A streptococcus) of different erythromycin resistance phenotypes were collected from all over Finland in 1994 and 1995 and studied; they were evaluated for their susceptibilities to 14 antimicrobial agents (396 isolates) and the presence of different erythromycin resistance genes (45 isolates). The erythromycin-resistant isolates with the macrolide-resistant but lincosamide- and streptogramin B-susceptible phenotype (M phenotype) were further studied for their plasmid contents and the transferability of resistance genes. Resistance to antimicrobial agents other than macrolides, clindamycin, tetracycline, and chloramphenicol was not found. When compared to our previous study performed in 1990, the rate of resistance to tetracycline increased from 10 to 93% among isolates with the inducible resistance (IR) phenotype of macrolide, lincosamide, and streptogramin B (MLSB) resistance. Tetracycline resistance was also found among 75% of the MLSB-resistant isolates with the constitutive resistance (CR) phenotype. Resistance to chloramphenicol was found for the first time in S. pyogenes in Finland; 3% of the isolates with the IR phenotype were resistant. All the chloramphenicol-resistant isolates were also resistant to tetracycline. Detection of erythromycin resistance genes by PCR indicated that, with the exception of one isolate with the CR phenotype, all M-phenotype isolates had the macrolide efflux (mefA) gene and all the MLSB-resistant isolates had the erythromycin resistance methylase (ermTR) gene; the isolate with the CR phenotype contained the ermB gene. No plasmid DNA could be isolated from the M-phenotype isolates, but the mefA gene was transferred by conjugation.

Streptococcus pyogenes isolated in Portugal: macrolide resistance phenotypes and correlation with T types. Portuguese Surveillance Group for the Study of Respiratory Pathogens.

From January 1998 to June 1999, 302 clinical isolates of Streptococcus pyogenes were collected from 10 microbiology laboratories in Portugal. All strains were highly sensitive to penicillin (MIC90 = 0.012 mg/liter). The prevalence of erythromycin resistance was 35.8% and of tetracycline resistance 41.4%. The majority (79.6 %) of erythromycin-resistant strains were of the MLSB constitutive resistance (CR) phenotype with high-level resistance to erythromycin (MIC90 >256 mg/liter) and to clindamycin (MIC90 >256 mg/liter), 16.7% showed the M phenotype with low-level erythromycin-resistance (MIC90 = 24 mg/liter) and susceptibility to clindamycin, and four isolates showed a phenotype characterized by low-level erythromycin resistance (MIC90 = 8 mg/liter) and high-level clindamycin resistance (MIC90 >256 mg/liter), not previously described. Erythromycin resistance was not associated with invasive strains. Only minor discrepancies between disk diffusion and E-test methods were observed. T serotyping was very useful for the epidemiological characterization of the strains. The most prevalent T types were T1, T4, T9, T12, T13, and T28. A statistically significant association with resistance patterns was found: T12 with erythromycin resistance MLS(B) CR phenotype (p< 0.001), T4 with erythromycin resistance M phenotype (p<0.001), and T13 with tetracycline resistance (p<0.01). Because of the high prevalence of resistance, careful surveillance of S. pyogenes isolates in Portugal is essential, routine antimicrobial susceptibility testing in clinical microbiology laboratories should be strongly encouraged, antibiotic prescription should be reviewed, and macrolides should no longer be used in the empirical therapy of acute pharyngitis.

Antibiotic susceptibility of group A streptococci in 2 Italian cities: Milano and Catania.

Resistance to macrolides has increasingly been reported for Group A streptococci. In this study, the in vitro antibiotic susceptibility pattern of 305 clinical isolates of S. pyogenes was determined. Strains were isolated during 1996 from pharyngeal swabs of children with uncomplicated pharyngitis living in 2 Italian cities: Milano and Catania, situated in the North and South of Italy, respectively. All isolates were found to be fully susceptible to penicillin and other beta-lactam agents tested. Susceptibility to macrolides differed markedly between the two centers with relatively high resistance rates to erythromycin being observed in Milano (30%) as compared to Catania (3%). Resistance to erythromycin was always crossed with that of the other 14- and 15-membered macrolides tested. However, resistance to josamycin and clindamycin was generally found in approximately 25% of the erythromycin-resistant (ER) strains. The erythromycin-resistant isolates from Milano and Catania (58 strains) were further subdivided into the three previously described resistance phenotypes: constitutive, inducible, and novel resistance phenotypes. The novel resistance phenotype accounted for 58% of all resistant strains, while 17% and 26% were found to be of the inducible and constitutive resistance phenotypes. Strains of the novel resistance phenotype were characterized by lower MIC values (MIC90 = 16 mg/L) to 14 and 15 carbon atom macrolides as compared to the other two phenotypes (MIC90 > 128 mg/L), and retained susceptibility to clindamycin and to josamycin, a 16 carbon atom macrolide. Resistance to tetracyclines was found in 25% to 36% of the ER isolates as compared to 2% to 10% of the susceptible strains. In particular, resistance to this agent was more commonly associated to isolates belonging to the novel and constitutive resistance phenotypes. MIC values for chloramphenicol in all isolates were within the susceptible or intermediate range; decreased susceptibility to this agent did not appear to be associated with erythromycin resistance.