Inducible Clindamycin Resistance and Biofilm Production among Staphylococci Isolated from Tertiary Care Hospitals in Nepal.
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ABSTRACT: Resistance to antibiotics, biofilm formation and the presence of virulence factors play important roles in increased mortality associated with infection by staphylococci. The macrolide lincosamide streptogramin B (MLSB) family of antibiotics is commonly used to treat infections by methicillin-resistant isolates. Clinical failure of clindamycin therapy has been reported due to multiple mechanisms that confer resistance to MLSB. This study aims to find the incidence of different phenotypes of MLSB resistance and biofilm production among staphylococci. A total of 375 staphylococci were isolated from different clinical samples, received from two tertiary care hospitals in Nepal. Methicillin resistance was detected by cefoxitin disc diffusion method and inducible clindamycin resistance by D test, according to CLSI guidelines. Biofilm formation was detected by the tissue culture plate method and PCR was used to detect ica genes. Of the total staphylococci isolates, 161 (42.9%) were Staphylococcus aureus, with 131 (81.4%) methicillin-resistant strains, and 214 (57.1%) isolates were coagulase-negative staphylococci, with 143 (66.8%) methicillin-resistant strains. The overall prevalence of constitutive MLSB (cMLSB) and inducible MLSB (iMLSB) phenotypes was 77 (20.5%) and 87 (23.2%), respectively. Both iMLSB and cMLSB phenotypes predominated in methicillin-resistant isolates. The tissue culture plate method detected biofilm formation in 174 (46.4%) isolates and ica genes in 86 (22.9%) isolates. Among biofilm producing isolates, cMLSB and iMLSB phenotypes were 35 (20.1%) and 27 (15.5%), respectively. The cMLSB and iMLSB were 11 (12.8%) and 19 (22.1%), respectively, in isolates possessing ica genes. Clindamycin resistance in the form of cMLSB and iMLSB, especially among MRSA, emphasizes the need for routine D tests to be performed in the lab.
Project description:Staphylococcus aureus, an important nosocomial pathogen, is frequently associated with infections in human. The management of the infections by it especially methicillin resistant ones is often difficult because methicillin resistant S. aureus is usually resistant to multiple antibiotics. Macrolide-lincosamide streptogramin B family of antibiotics is commonly used to treat such infections as an alternative to vancomycin.This study was conducted over the period of one and half year from November 2013-April 2015 in Microbiology laboratory of Nepal Medical College and Teaching Hospital, Kathmandu, Nepal to find the incidence of different phenotypes of MLSB resistance among S. aureus from clinical samples and their association with methicillin resistance. Two hundred seventy isolates of S. aureus were included in the study. Methicillin resistance was detected by cefoxitin disc diffusion method and inducible clindamycin resistance by erythromycin and clindamycin disc approximation test (D-test).Of the 270 clinical isolates of S. aureus, 25.1% (68/270) were MRSA. Erythromycin and clindamycin resistance was seen in 54.4% (147/270) and 41.8% (113/270) isolates respectively. Resistance to erythromycin and clindamycin were higher in MRSA as compared to MSSA (erythromycin-resistance: 88.2% Vs 39.1% and clindamycin-resistance: 79.4% Vs 41.8%). The overall prevalence of iMLSB and cMLSB phenotype was 11.48% (31/270) and 29.25% (79/270) respectively. Both iMLSB and cMLSB phenotypes predominated in MRSA strains.Detection rate of MRSA in our study shows the necessity to improve in healthcare practices and to formulate new policy for the control of MRSA infections. Clindamycin resistance in the form of iMLSB and cMLSB especially among MRSA emphasizes the need of D-test to be performed routinely in our set up while using clindamycin as an alternative choice to anti-staphylococcal antibiotics like vancomycin and linezolid in the treatment of staphylococcal infections.
Project description:Staphylococcus epidermidis strains isolated from nosocomial infections represent a serious problem worldwide. In various Mexican states several reports have shown isolates from hospitals with antibiotic resistance to methicillin. In Mexico City, there is scarce information on staphylococcal infections in hospitals. Here, our research findings are shown in a four-year period study (2006-2010) for Staphylococcus epidermidis strains. Susceptibility and/or resistance to antibiotics in SE strains were assessed by phenotypic and molecular methods as mecA gene by PCR, as well as the correlation with biofilm production for these isolates and the relationship to the infection site. Out of a total of 161 (66%) negative biofilm SE strains, just 103 (64%) SE strains were confirmed as MRSE by PCR to mecA gene. From 84 (34%) positive biofilm SE strains, 76 (91%) were confirmed as MRSE by PCR to mecA gene. Higher percentages of resistance to antibiotics and higher number of resistance markers were found in biofilm-forming clinical strains (9 to 14) than non-biofilm-forming SE strains (3 to 8). These research findings represent a guide to establish infection control programs for this hospital.
Project description:BackgroundLinezolid is one of the most effective treatments against Gram-positive pathogens. However, linezolid-resistant/intermediate strains have recently emerged in worldwide. The purpose of this study was to analyse the prevalence and resistance mechanisms of linezolid-resistant/intermediate staphylococci and enterococci in Shanghai, China.ResultsThirty-two linezolid-resistant/intermediate strains, including 14 Staphylococcus capitis, three Staphylococcus aureus, 14 Enterococcus faecalis and one Enterococcus faecium clinical isolates, were collected in this study which displayed linezolid MICs of 8 to 512 μg/ml, 8-32 μg/ml, 4-8 μg/ml and 4 μg/ml, respectively. All linezolid-resistant S. capitis isolates had a novel C2131T mutation and a G2603T mutation in the 23S rRNA region, and some had a C316T (Arg106Cys) substitution in protein L4 and/or harboured cfr. Linezolid-resistant S. aureus isolates carried a C389G (Ala130Gly) substitution in protein L3, and/or harboured cfr. The cfr gene was flanked by two copies of the IS256-like element, with a downstream orf1 gene. Linezolid-resistant/intermediate enterococci lacked major resistance mechanisms. The semi-quantitative biofilm assay showed that 14 linezolid-resistant E. faecalis isolates produced a larger biofilm than linezolid-susceptible E. faecalis strains. Transmission electron microscopy showed the cell walls of linezolid-resistant/intermediate strains were thicker than linezolid-susceptible strains.ConclusionOur data indicated that major resistance mechanisms, such as mutations in 23S rRNA and ribosomal proteins L3 and L4, along with cfr acquisition, played an important role in linezolid resistance. Secondary resistance mechanisms, such as biofilm formation and cell wall thickness, should also be taken into account.
Project description:Staphylococcus aureus, a notorious human pathogen, is a major cause of the community as well as healthcare associated infections. It can cause a diversity of recalcitrant infections mainly due to the acquisition of resistance to multiple drugs, its diverse range of virulence factors, and the ability to produce biofilm in indwelling medical devices. Such biofilm associated chronic infections often lead to increase in morbidity and mortality posing a high socio-economic burden, especially in developing countries. Since biofilm formation and antibiotic resistance function dependent on each other, detection of biofilm expression in clinical isolates would be advantageous in treatment decision. In this premise, we attempt to investigate the biofilm formation and its association with antibiotic resistance in clinical isolates from the patients visiting tertiary health care hospitals in Nepal. Bacterial cells isolated from clinical samples identified as S. aureus were examined for in-vitro biofilm production using both phenotypic and genotypic assays. The S. aureus isolates were also examined for susceptibility patterns of clinically relevant antibiotics as well as inducible clindamycin resistance using standard microbiological techniques and D-test, respectively. Among 161 S. aureus isolates, 131 (81.4%) were methicillin resistant S. aureus (MRSA) and 30 (18.6%) were methicillin sensitive S. aureus (MSSA) strains. Although a majority of MRSA strains (69.6%) showed inducible clindamycin resistance, almost all isolates (97% and 94%) were sensitive toward chloramphenicol and tetracycline, respectively. Detection of in vitro production of biofilm revealed the association of biofilm with methicillin as well as inducible clindamycin resistance among the clinical S. aureus isolates.
Project description:IntroductionHospital based delivery has been an expensive experience for poor households because of hidden costs which are usually unaccounted in hospital costs. The main aim of this study was to estimate the hidden costs of hospital based delivery and determine the factors associated with the hidden costs.MethodsA hospital based cross-sectional study was conducted among 384 post-partum mothers with their husbands/house heads during the discharge time in Manipal Teaching Hospital and Western Regional Hospital, Pokhara, Nepal. A face to face interview with each respondent was conducted using a structured questionnaire. Hidden costs were calculated based on the price rate of the market during the time of the study.ResultsThe total hidden costs for normal delivery and C-section delivery were 243.4 USD (US Dollar) and 321.6 USD respectively. Of the total maternity care expenditures; higher mean expenditures were found for food & drinking (53.07%), clothes (9.8%) and transport (7.3%). For postpartum women with their husband or house head, the total mean opportunity cost of "days of work loss" were 84.1 USD and 81.9 USD for normal delivery and C-section respectively. Factors such as literate mother (p = 0.007), employed house head (p = 0.011), monthly family income more than 25,000 NRs (Nepalese Rupees) (p = 0.014), private hospital as a place of delivery (p = 0.0001), C-section as a mode of delivery (p = 0.0001), longer duration (>5days) of stay in hospital (p = 0.0001), longer distance (>15km) from house to hospital (p = 0.0001) and longer travel time (>240 minutes) from house to hospital (p = 0.007) showed a significant association with the higher hidden costs (>25000 NRs).ConclusionExperiences of hidden costs on hospital based delivery and opportunity costs of days of work loss were found high. Several socio-demographic factors, delivery related factors (place and mode of delivery, length of stay, distance from hospital and travel time) were associated with hidden costs. Hidden costs can be a critical factor for many poor and remote households who attend the hospital for delivery. Current remuneration (10-15 USD for normal delivery, 30 USD for complicated delivery and 70 USD for caesarean section delivery) for maternity incentive needs to account the hidden costs by increasing it to 250 USD for normal delivery and 350 USD for C-section. Decentralization of the obstetric care to remote and under-privileged population might reduce the economic burden of pregnant women and can facilitate their attendance at the health care centers.
Project description:: Staphylococcus aureus (S. aureus) is one of the most clinically important zoonotic pathogens, but an understanding of the prevalence, biofilm formulation ability, virulence, and antimicrobial resistance genes of S. aureus from veterinary hospitals is lacking. By characterizing S. aureus in different origins of veterinary hospitals in Guangzhou, China, in 2019, we identified with the presence of S. aureus in pets (17.1%), veterinarians (31.7%), airborne dust (19.1%), environmental surfaces (4.3%), and medical device surfaces (10.8%). Multilocus sequence typing (MLST) and Staphylococcus protein A (spa) typing analyses demonstrated methicillin-sensitive S. aureus (MSSA) ST398-t571, MSSA ST188-t189, and methicillin-resistant S. aureus (MRSA) ST59-t437 were the most prevalent lineage. S. aureus with similar pulsed-field gel electrophoresis (PFGE) types distributed widely in different kinds of samples. The crystal violet straining assays revealed 100% (3/3) of MRSA ST59 and 81.8% (9/11) of MSSA ST188 showed strong biofilm formulation ability, whereas other STs (ST1, ST5, ST7, ST15, ST88, ST398, ST3154 and ST5353) showed weak biofilm production ability. Polymerase chain reaction (PCR) confirmed the most prevalent leucocidin, staphylococcal enterotoxins, ica operon, and adhesion genes were lukD-lukE (49.0%), sec-sel (15.7%), icaA-icaB-icaC-icaR (100.0%), and fnbB-cidA-fib-ebps-eno (100.0%), respectively. Our study showed that the isolates with strong biofilm production ability had a higher prevalence in clfA, clfB, fnbA and sdrC genes compared to the isolates with weak biofilm production ability. Furthermore, 2 ST1-MRSA isolates with tst gene and 1 ST88-MSSA isolate with lukS/F-PV gene were detected. In conclusion, the clonal dissemination of S. aureus of different origins in veterinary hospitals may have occurred; the biofilm production capacity of S. aureus is strongly correlated with ST types; some adhesion genes such as clfA, clfB, fnbA, and sdrC may pose an influence on biofilm production ability and the emergence of lukS/F-PV and tst genes in S. aureus from veterinary hospitals should raise our vigilance.
Project description:BackgroundCoagulase-negative staphylococci (CNS) survive as commensals of skin, anterior nares and external canals of human and were regarded as non-infectious pathogens. However, they are emerging as a major cause of nosocomial infectious due to their ability to form biofilms and high resistance to several classes of antibiotics. This study examines the biofilm forming abilities of 214 clinical CNS isolates using phenotypic and genotypic methods, and determines their antibiotic susceptibility patterns.MethodsA total of 214 clinical isolates collected from different clinical samples were identified as CNS and their antibiotic susceptibility determined by CLSI guidelines. The biofilm forming ability of all isolates was determined by three phenotypic methods; Congo red agar (CRA) method, tube adherence method (TM) and tissue culture plate (TCP) method and by genotypic method for the detection of icaAD genes.ResultsAmong all the isolates, S. epidermidis (57.5%) was found the most frequently, followed by S. saprophyticus (18.7%), S. haemolyticus (11.2%), S. hominis (7%), and S. capitis (5.6%). Antibiotic susceptibility pattern demonstrated 91.6% isolates were resistant to penicillin and 66.8% to cefoxitin while 91.1% isolates were susceptible to chloramphenicol. Constitutive and inducible clindamycin resistant phenotype as measured by D-test was seen among 28% and 14.5% of isolates respectively. Tissue culture plate method detected biofilm production in 42.1% isolate followed by 31.8% through tube method while 20.1% isolates were found to produce slime in Congo red agar method. The genotypic assay revealed presence of icaA and icaD genes in 19.2% isolates.ConclusionThe study shows a high prevalence of biofilm formation and inducible clindamycin resistance in CNS isolates, indicating the importance of in-vitro biofilm production test and D-test in routine laboratory diagnostics. Implementation of efficient diagnostic techniques for detection of biofilm production in clinical samples can help manage staphylococcal infections and minimize risks of treatment failures in hospitals.
Project description:The presence of biofilms can negatively affect several different areas, such as the food industry, environment, and biomedical sectors. Conditions under which bacteria grow and develop, such as temperature, nutrients, and pH, among others, can largely influence biofilm production. Staphylococcus species survive in the natural environment due to their tolerance to a wide range of temperatures, dryness, dehydration, and low water activity. Therefore, we aimed to evaluate the influence of external environmental factors on the formation of biofilm of staphylococci isolated from hospital wastewater and surface waters. We investigated the biofilm formation of methicillin-resistant and -susceptible S. aureus (MRSA and MSSA) and coagulase-negative staphylococci (CoNS) under various temperatures, pH values, salt concentrations, glucose concentrations, and under anaerobic and aerobic conditions. CoNS had the ability to produce more biofilm biomass than MSSA and MRSA. All environmental factors studied influenced the biofilm formation of staphylococci isolates after 24 h of incubation. Higher biofilm formation was achieved at 4% of NaCl and 0.5% of glucose for MSSA and CoNS, and 1% of NaCl and 1.5% of glucose for MRSA isolates. Biofilm formation of isolates was greater at 25 °C and 37 °C than at 10 °C and 4 °C. pH values between 6 and 8 led to more robust biofilm formation than pH levels of 9 and 5. Although staphylococci are facultative anaerobes, biofilm formation was higher in the presence of oxygen. The results demonstrated that multiple environmental factors affect staphylococci biofilm formation. Different conditions affect differently the biofilm formation of MRSA, MSSA, and CoNS strains.
Project description:This study compared the molecular characteristics of vancomycin-resistant Enterococcus faecium (VREF) isolates recovered from 20 non-tertiary-care hospitals (36 isolates) and three tertiary-care hospitals (26 isolates) in diverse geographical areas of Korea from October 2010 to April 2011. All isolates carried the vanA gene only, but 42% and 73% of non-tertiary and tertiary-care isolates expressed the VanB phenotype (teicoplanin minimum inhibitory concentration ⩽16 μg/ml). All isolates harboured insertion sequences, IS1542 and IS1216V, within Tn1546. The isolates from tertiary-care hospitals tended to have reduced Tn1546 lengths by deletion of sequences adjacent to IS elements. Multilocus sequence typing revealed eight sequence types within clonal complex 17 (CC17), but DNA fingerprinting by rep-PCR did not show clonal relatedness between the intra- and inter-hospital isolates. These results suggest that vanA, which has prevailed in tertiary-care hospitals of Korea since the 1990s, had been transferred horizontally to non-tertiary-care hospitals while the genetic rearrangement driven by evolutionary adaptation to adverse environments may have occurred in tertiary-care hospitals.
Project description:Methicillin-resistant coagulase negative staphylococci (MR-CoNS) are the major cause of infectious diseases because of their potential ability to form biofilm and colonize the community or hospital environments. This study was designed to investigate the biofilm producing ability, and the presence of mecA, icaAD, bap and fnbA genes in MR-CoNS isolates. The MR-CoNS used in this study were isolated from various samples of community environment and five wards of hospital environments, using mannitol salt agar (MSA) supplemented with 4 μg/ml of oxacillin. The specie level of Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus hominis and Staphylococcus warneri was identified by specific primers of groESL (S. haemolyticus), rdr (S. epidermidis) and nuc (S. hominis and S. warneri). The remainder isolates were identified by tuf gene sequencing. Biofilm production was determined using Congo red agar (CRA) and Microtiter plate (MTP) assay. The mecA and biofilm associated genes (icaAD, fnbA and bap) were detected using PCR method. From the 558 samples from community and hospital environments, 292 MR-CoNS were isolated (41 from community environments, and 251 from hospital environments). S. haemolyticus (41.1%) and S. epidermidis (30.1%) were the predominant species in this study. Biofilm production was detected in 265 (90.7%) isolates by CRA, and 260 (88.6%) isolates were detected by MTP assay. The staphylococci isolates derived from hospital environments were more associated with biofilm production than the community-derived isolates. Overall, the icaAD and bap genes were detected in 74 (29.5%) and 14 (5.6%) of all isolates from hospital environments. When tested by MTP, the icaAD gene from hospital environment isolates was associated with biofilm biomass. No association was found between bap gene and biofilm formation. The MR-CoNS isolates obtained from community environments did not harbor the icaAD and bap genes. Conversely, fnbA gene presented in MR-CoNS isolated from both community and hospital environments. The high prevalence of biofilm producing MR-CoNS strains demonstrated in this study indicates the persisting ability in environments, and is useful in developing prevention strategies countering the spread of MR-CoNS.