Macrolide Lincosamide Research Articles

Manure and Doxycycline Affect the Bacterial Community and Its Resistome in Lettuce Rhizosphere and Bulk Soil.

Manure application to agricultural soil introduces antibiotic residues and increases the abundance of antibiotic-resistant bacteria (ARB) carrying antibiotic resistance genes (ARGs), often located on mobile genetic elements (MGEs). The rhizosphere is regarded as a hotspot of microbial activity and gene transfer, which can alter and prolong the effects of organic fertilizers containing antibiotics. However, not much is known about the influence of plants on the effects of doxycycline applied to soil via manure. In this study, the effects of manure spiked with or without doxycycline on the prokaryotic community composition as well as on the relative abundance of ARGs and MGEs in lettuce rhizosphere and bulk soil were investigated by means of a polyphasic cultivation-independent approach. Samples were taken 42 days after manure application, and total community DNA was extracted. Besides a pronounced manure effect, doxycycline spiking caused an additional enrichment of ARGs and MGEs. High-throughput quantitative PCR revealed an increase in tetracycline, aminoglycoside, and macrolide–lincosamide–streptogramin B (MLSB) resistance genes associated with the application of manure spiked with doxycycline. This effect was unexpectedly lower in the rhizosphere than in bulk soil, suggesting a faster dissipation of the antibiotic and a more resilient prokaryotic community in the rhizosphere. Interestingly, the tetracycline resistance gene tetA(P) was highly enriched in manure-treated bulk soil and rhizosphere, with highest values observed in doxycycline-treated bulk soil, concurring with an enrichment of Clostridia. Thus, the gene tetA(P) might be a suitable marker of soil contamination by ARB, ARGs, and antibiotics of manure origin. These findings illustrate that the effects of manure and doxycycline on ARGs and MGEs differ between rhizosphere and bulk soil, which needs to be considered when assessing risks for human health connected to the spread of ARGs in the environment.

Streptococcus agalactiae from Ethiopian pregnant women; prevalence, associated factors and antimicrobial resistance: alarming for prophylaxis

BackgroundMaternal Streptococcus agalactiae (Group B Streptococcus, GBS) colonization rates and its antibiotic resistance patterns provide important information useful in guiding prevention strategies. There is a paucity of evidence about GBS in the Amhara National Regional State, Ethiopia.ObjectiveTo determine colonization prevalence, associated risk factors, and antibiotics resistance including inducible clindamycin resistance patterns of GBS among Ethiopian pregnant women.MethodsA prospective cross-sectional study was conducted from 1st December 2016 to 30th November 2017 at the University of Gondar Referral hospital delivery ward. Combined recto-vaginal swabs were collected from 385 pregnant women and analyzed at the University of Gondar Bacteriology Laboratory by using LIM broth and 5% defibrinated sheep blood agar culture methods. Isolates were identified by using colony morphology, gram reaction, hemolysis, and CAMP test. Antibiotic susceptibility test was done using the disc diffusion method. Double disc diffusion method was used to identify inducible clindamycin resistance isolates. Data were analyzed by SPSS version 20 software. p ≤ 0.05 was considered as statistically significant.ResultsThe overall prevalence of maternal GBS colonization was 25.5% (95% CI 21–29.5%). Experiencing meconium stained amniotic fluid (AOR = 3.018, 95% CI 1.225, 7.437), and longer duration of premature rupture of membrane (AOR = 1.897, 95% CI 1.014, 3.417) were statistically significant to maternal colonization. Furthermore, GBS resistant to 0 (8.2%), 1 (25.5%) and 3 (39.8%) or more antibiotics were identified. A D-test showed 15.2% inducible clindamycin resistant GBS. Constitutive macrolide lincosamide–streptograminB, L-, and M-phenotypes were also detected.ConclusionsMaternal GBS colonization rate in this study was higher compared to the previous reports in Ethiopia. This much prevalence and antibiotics resistance results are the clue to which attention shall be given to this bacterium during management of pregnant women and the newborns.

Prevalence, multidrug resistance and molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) in retail meat from Punjab, India

This study reports the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in retail meat from Punjab, India. Classical microbiological methods were applied to isolate and identify S. aureus isolates. Isolates also underwent Etest. PCR and sequencing were used to identify and characterise antimicrobial resistance genes. MLST, SCCmec and spa typing were performed. A total of 408 meat and 101 swab samples were processed for S. aureus isolation. Phenotypic resistance was highest to penicillin (90.97%), followed by ciprofloxacin (61.80%), tetracycline (45.14%) and erythromycin (11.11%). Isolates from chicken samples showed significantly higher MICs for tetracycline than chevon and pork samples and significantly higher MICs for trimethoprim/sulfamethoxazole and gentamicin than chevon and swab samples (P<0.05). No isolates were phenotypically resistant to vancomycin (MICs of 0.5-2μg/mL). Most isolates (52.78%, 95% CI 44.63-60.93%) were multidrug-resistant and carried resistance genes to penicillin (blaZ), oxacillin (mecA), gentamicin (aacA-aphD), erythromycin (ermB, ermC) and tetracycline (tetK, tetL, tetM). MRSA was only found in chicken samples (2.72%; 4/147). Seven S. aureus (5.07%) were borderline oxacillin-resistant (MIC range 4-8μg/mL). All MRSA were SCCmecV-pvl+-t442, among which three isolates were ST5. Their genotype was mecA+, blaZ+, aacA-aphD+, tetK+, ermC+/-. Among the erythromycin-resistant isolates, 25% were MRSA, of which 12.5% isolates expressed an inducible macrolide-lincosamide-streptogramin B (iMLSB) phenotype. These data confirm the presence of ST5-t442-MRSA-SCCmecV-pvl+ and iMLSB MRSA in meat samples, indicating a potential role of meat in the dissemination of multidrug-resistant S. aureus strains and successful MRSA lineages in Punjab.

Combined impact of fishmeal and tetracycline on resistomes in mariculture sediment

Mariculture sediment has been recognized as a major contributor of environmental antibiotic resistance genes (ARGs), which are challenging the treatment of infections worldwide. Both antibiotics and fishmeal are used in aquaculture, and each has the potential to facilitate ARG dissemination, however their combined impact on the sediment resistome and their relative contribution remain unclear. In this study, microcosms were exposed to varying concentrations of tetracycline with or without fishmeal (0.1% wt/wt) for 14 days. Sediment genomic DNA was analyzed using high throughput quantitative PCR and 16S rRNA gene amplicon sequencing to compare the contribution of fishmeal and tetracycline to antibiotic resistomes and bacterial communities in mariculture sediment. Sixty-seven ARGs were detected potentially correlating to resistance for several major antibiotics. Fishmeal, but not the dose of tetracycline, contributed to the significant increase of both ARG abundance and diversity in the sediment. Based on principle coordinate analysis and hierarchical clustering, ARGs were clustered into two groups depending on whether fishmeal was added. Aminoglycoside, macrolide–lincosamide–streptogramin b (MLSb) and tetracycline resistance genes were the most abundant when fishmeal was used, while a significant increase in mobile genetic element (MGE) abundance was also detected (P < 0.05). Meanwhile, bacterial community structures were detected with distinct patterns between the two groups (Adonis, P < 0.05). Using the Mantel test and partial least squares path modeling, we identified that sediment resistomes were significantly correlated with microbial community structures (P < 0.05) which were mainly driven by nutrients in fishmeal. Together our findings suggested that fishmeal plays a more important role than tetracycline in proliferation of ARGs in mariculture sediment. This study may provide new insights into the mitigation of ARG propagation in mariculture operations.

Prevalence of Genotypes That Determine Resistance of Staphylococci to Macrolides and Lincosamides in Serbia.

Macrolides, lincosamides, and streptogramins (MLS) resistance genes are responsible for resistance to these antibiotics in Staphylococcus infections. The purpose of the study was to analyze the distribution of the MLS resistance genes in community- and hospital-acquired Staphylococcus isolates. The MLS resistance phenotypes [constitutive resistance to macrolide–lincosamide–streptogramin B (cMLSb), inducible resistance to macrolide–lincosamide–streptogramin B (iMLSb), resistance to macrolide/macrolide–streptogramin B (M/MSb), and resistance to lincosamide–streptogramin A/streptogramin B (LSa/b)] were determined by double-disc diffusion method. The presence of the MLS resistance genes (ermA, ermB, ermC, msrA/B, lnuA, lnuB, and lsaA) were determined by end-point polymerase chain reaction in 179 isolates of staphylococci collected during 1-year period at the Center for Microbiology of Public Health Institute in Vranje. The most frequent MLS phenotype among staphylococcal isolates, both community-acquired and hospital-acquired, was iMLSb (33.4%). The second most frequent was M/MSb (17.6%) with statistically significantly higher number of hospital-acquired staphylococcal isolates (p < 0.05). MLS resistance was mostly determined by the presence of msrA/B (35.0%) and ermC (20.8%) genes. Examined phenotypes were mostly determined by the presence of one gene, especially by msrA/B (26.3%) and ermC (14.5%), but 15.6% was determined by a combination of two or more genes. M/MSb phenotype was the most frequently encoded by msrA/B (95.6%) gene, LSa/b phenotype by lnuA (56.3%) gene, and iMLSb phenotype by ermC (29.4%) and ermA (25.5%) genes. Although cMLSb phenotype was mostly determined by the presence of ermC (28.9%), combinations of two or more genes have been present too. This pattern was particularly recorded in methicillin-resistant Staphylococcus aureus (MRSA) (58.3%) and methicillin-resistant coagulase-negative staphylococci (MRCNS) (90.9%) isolates with cMLSB phenotype. The msrA/B gene and M/MSb phenotype were statistically significantly higher in hospital-acquired than community-acquired staphylococci strains (p < 0.05). There are no statistically significant differences between staphylococci harboring the rest of MLS resistance genes acquired in community and hospital settings (p > 0.05). The prevalence of iMLSb phenotypes may change over time, so it is necessary to perform periodic survey of MLS resistance phenotypes, particularly where the D-test is not performed routinely.

Prevalence of methicillin resistance and macrolide–lincosamide–streptogramin B resistance in Staphylococcus haemolyticus among clinical strains at a tertiary-care hospital in Thailand

Staphylococcus spp. is a major cause of nosocomial infection and sepsis. However, increasing drug resistance is becoming a challenge to microbiologists. The purpose of this study was to identify and determine antimicrobial resistance phenotypes and drug resistance genes of clinical coagulase-negative staphylococci (CoNS) isolates at Mae Sot Hospital in Tak province, Thailand. A total of 229 CoNS isolates were collected from clinical specimens during two periods in 2014 and in 2015. Staphylococcus haemolyticus was the most prevalent species (37.55%), followed by S. epidermidis (21.83%), S. saprophyticus (11.79%) and S. hominis (11.35%) respectively. The remaining 17.48% of the organisms comprised S. capitis, S. arlettae, S. cohnii, S. equorum, S. xylosus, S. warneri, S. sciuri, S. pettenkoferi, S. kloosii and S. lugdunensis. Methicillin-resistant CoNS (MRCoNS), containing the mecA gene, were detected in 145 of 229 isolates, mostly found in S. haemolyticus and S. epidermidis. In addition, the differentiation of their macrolide–lincosamide–streptogramin B (MLSB) resistance phenotypes was determined by the D-test and corresponding resistance genes. Among 125 erythromycin-resistant CoNS, the prevalence of constitutive type of MLSB, inducible clindamycin resistance and macrolide–streptogramin B resistance phenotypes were 72, 13.60 and 14.40% respectively. These phenotypes were expressed in 80% of MRCoNS strains. In addition, the ermC gene (79.20%) was found to be more prevalent than the ermA gene (22.40%), especially among MRCoNS. These results indicate that CoNS may play an important role in spreading of drug resistance genes. More attention to these organisms in surveillance and monitoring programs is needed.

Prevalence of macrolide-lincosamide resistance and multidrug resistance phenotypes in streptococcal isolates causing infections in European hospitals: Evaluation of the in vitro activity of oritavancin and comparator agents.

The aim of this study was to evaluate the prevalence of resistance to erythromycin alone (M) and to erythromycin and clindamycin (cMLSB) as well as multidrug resistance (MDR) phenotypes (resistance to at least three classes of drugs) among clinical enterococci from European countries and adjacent geographic regions. The in vitro activity of oritavancin against these isolates was also evaluated. A total of 2569 streptococci collected from 12 European countries as well as Russia, Turkey and Israel were included. A total of 9.8%, 8.1% and 6.4% of β-haemolytic streptococci (BHS) displayed M, cMLSB and MDR phenotypes, respectively. Oritavancin (99.4-100.0% susceptible) demonstrated modal minimum inhibitory concentration (MIC) (0.03mg/L) and MIC50 (0.03mg/L) values that were the same for all BHS or subsets, including MDR. The oritavancin MIC50 value of 0.06mg/L against Streptococcus dysgalactiae was similar to those of daptomycin and penicillin (MIC50≤0.06mg/L for both). Among viridans group streptococci (VGS), 28.3%, 12.7% and 11.6% showed M, cMLSB and MDR phenotypes, respectively. Oritavancin (99.9-100.0% susceptible; MIC50/90, ≤0.008/0.06mg/L) exhibited potent in vitro activity against VGS and resistant subsets, as did vancomycin (MIC50/90, 0.5/0.5-1mg/L), daptomycin (MIC50/90, 0.25-0.5/0.5-1mg/L) and linezolid (MIC50/90, 0.5-1/1mg/L). In conclusion, rates of resistance phenotypes were higher in VGS than BHS. Oritavancin demonstrated in vitro potencies that were similar to or greater than those of comparators against this recent collection of streptococci, including drug-resistant subsets, from European and adjacent countries.

The sensitivity to antibiotics of strains of group B streptococcus isolates from pregnant women in Belgrade

Introduction: Group B streptococcus (GBS) is a significant human pathogen. GBS colonizes the vagina and it is one of the most important causes of early neonatal sepsis and meningitis. In many countries, screening of pregnant women and intrapartal use of antibiotics are common practice. Macrolide and lincosamide resistant strains of GBS are a significant problem, because these antibiotics are the second line therapy in case of penicillin allergy. Aim: Our aim was to investigate the frequency of antibiotic resistant strains of GBS and to detect macrolide resistance phenotypes in GBS strains obtained from pregnant women in Belgrade. Material and Methods: 105 GBS isolates were obtained from vaginal swabs of pregnant women attending two Gynecology and Obstetrics Centers in Belgrade. The isolates were tested for antimicrobial susceptibility pattern and D test were performed on Mueller Hinton agar. Results: Macrolide and lincosamide resistance was found in 30.4 %, and 23.8 % of isolates, respectively. There was a high frequency of tetracycline resistant strains (88.6 %). Most frequent macrolide resistant phenotype was iMLSb (macrolide and inducibile lincosamide resistance) (62.4%). Conclusion: The results of our study indicate that there is a high level of macrolide resistance among GBS isolates in Serbia and the active surveillance is needed.

Inducible clindamycin resistance among clinical isolates of Staphylococcus aureus

Objective: The aim of the study was to detect the presence of inducible clindamycin resistance among clinical isolates of Staphylococcus aureus. Background: The resistance to antimicrobial agents among staphylococci is an increasing problem. This has led to renewed interest in the usage of macrolide–lincosamide–streptogramin B (MLSB) antibiotics to treat S. aureus infections. The resistance to macrolide can be mediated by the msrA gene coding for efflux mechanism or by the erm gene encoding for enzymes that confer inducible or constitutive resistance to MLSB antibiotics. Materials and methods: Ninety staphylococcal isolates were collected and were tested for antibiotic susceptibility using the disk diffusion method. Inducible resistance to clindamycin in S. aureus was tested by using the double disk diffusion test as per CLSI guidelines. Results: Inducible MLSBresistance (EryRClinInd) was seen in 7/90 (7.7%) isolates, the constitutive phenotype of MLSBresistance (EryRClinR) was detected in 6/90 (6.6%) isolates, and the MSBphenotype (EryRClinS) was seen in 34 (37.7%) isolates. In these isolates, inducible clindamycin resistance was detected in 4.3% (3/70) of S. aureus and 20% (4/20) of coagulase-negative staphylococci. Conclusion: This study showed that the double disk diffusion test should be used as a mandatory method in routine disk diffusion testing to detect inducible clindamycin resistance in staphylococci for the optimum treatment of patients.

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.

The Rate of Inducible MLSBResistance in the Methicillin‐Resistant Staphylococci Isolated From Clinical Samples

BackgroundStaphylococci are one of the most common pathogens in nasocomial and community‐acquired infections. Methicillin‐resistant staphylococci are known to be resistant against all beta‐lactam antibiotics. Therefore, non‐beta‐lactam antibiotics such as macrolide and lincosamides can be used. Resistance to those antibiotics may lead to therapeutic failure. The purpose of the present study was to determine the prevalence of macrolide–lincosamide–streptogramin B (MLSB) resistance by usingD‐test in staphylococcal isolates from various clinical samples.MethodsSeventy‐one methicillin‐resistant Staphylococcus isolates (sixS. aureus, 65 coagulase negative staphylococci) were included in this study. Staphylococci were identified with conventional methods. According to Clinical Laboratory Standards Institute (CLSI) criteria, susceptibility testing was performed by Kirby–Bauer disk diffusion method.ResultsOne of six (16.6%) methicillin‐resistantS. aureusisolates and 19 of 65 (29.2%) methicillin‐resistant coagulase‐negative staphylococci (MR‐CNS) were detected asD‐test positive. Twenty of 71 (28.1%) staphylococcal isolates detected asD‐test positive. Inducible clindamycin resistance was found at a higher rate in MR‐CNS.ConclusionSince the resistant community and hospital acquired staphylococcal infections have become a therapeutic problem, it is very important to detect MLSBresistance routinely in microbiology laboratories.D‐test is a cheap and reliable diagnostic method that can be performed in every laboratory.

Antimicrobial resistance and population structure of Staphylococcus aureus recovered from pigs farms

Staphylococcus aureus is a burden in human and veterinary medicine. During the last decade, an increasing number of studies reported the presence of livestock-associated methicillin-resistant S. aureus (LA-MRSA) clonal complex (CC) 398 in pigs. During 2013, a survey was performed in pig farms (n=328) randomly selected over Belgium, to monitor the current epidemiological situation of LA-MRSA among asymptomatic pigs and compare with former data to determine possible evolutions. Per farm, nose swabs were taken from 20 animals and pooled. MRSA was detected in 215 farms. Most isolates belonged to CC398 (n=211), and the remaining were ST9/t337 (n=1), ST80/t044 (n=2) and ST239/t4150 (n=1). A large diversity (n=19) of spa-types was found in the CC398 isolates. More than 90% of the isolates were non-wild type (NWT) to tetracycline and trimethoprim. NWT isolates were also found for ciprofloxacin (61.1%), clindamycin (64.4%), erythromycin (57.8%), kanamycin (43.1%) and gentamicin (45.5%). Microarray analysis showed that most CC398 isolates carried genes encoding resistance to tetracycline [tet(M)], macrolide–lincosamide–streptogramin group [erm(B), erm(C), lnu(A), vga(A)], aminoglycosides (aacA-aphD,aa dD, aphA3, sat) and/or phenicols (fexA). One CC398 isolate carried the multi-resistance gene cfr. The non-CC398 isolates carried virulence genes, as the egc-like cluster. The ST80 strain carried the Panton–Valentine leukocidin gene and corresponded to the community-acquired (CA-)MRSA ST80-IV European clone. The MRSA prevalence among pigs in Belgium remains similar to previous studies but a larger diversity in spa-types has been detected in this study. The recovery of CA-MRSA from livestock indicates that one should remain vigilant to the evolution of LA-MRSA in pigs.

Antimicrobial resistance, virulence factors and genetic lineages of hospital-onset methicillin-resistant Staphylococcus aureus isolates detected in a hospital in Zaragoza

IntroductionMRSA population dynamics is undergoing significant changes, and for this reason it is important to know which clones are circulating in our nosocomial environment. Materials and methodsA total of 118 MRSA isolates were collected from clinical samples from patients with previous hospital or healthcare contact (named as hospital-onset MRSA (HO-MRSA)) during a one year period. Susceptibility testing was performed by disk diffusion and microdilution. The presence of resistance genes and virulence factors were tested by PCR. All isolates were typed by SCCmec, spa and agr typing. PFGE and MLST were applied to a selection of them. ResultsEighty-three HO-MRSA isolates (70.3%) were resistant to any antibiotic included in the macrolide–lincosamide–streptogramin B group. Among these isolates, the M phenotype was the most frequent (73.5%). One hundred and seven of HO-MRSA isolates (90.7%) showed aminoglycoside resistance. The combination aac(6′)-Ie-aph(2″)-Ia+ant(4′)-Ia genes was the most frequent (22.4%). Tetracycline resistance rates in HO-MRSA isolates were low (3.4%), although a high level of mupirocin resistance was observed (25.4%). Most of the HO-MRSA isolates (approximately 90%) showed SCCmec type IVc and agr type II. Fifteen unrelated pulsotypes were identified. CC5 was the most prevalent (88.1%), followed by CC8 (5.9%), CC22 (2.5%), CC398 (2.5%) and CC1 (0.8%). ConclusionCC5/ST125/t067 lineage was the most frequent. This lineage was related to aminoglycoside resistance, and to a lesser extent, with macrolide resistance. The presence of international clones as EMRSA-15 (CC22/ST22), European clones as CC5/ST228, community clones related to CC1 or CC8 and livestock associated clones, as CC398, were observed in a low percentage.

Decreased Susceptibility to Macrolide-Lincosamide in Mycoplasma synoviae Is Associated with Mutations in 23S Ribosomal RNA.

The mechanism responsible for acquired decreased susceptibility to macrolides (14-membered erythromycin [Ery], 16-membered tylosin [Ty] and tilmicosin [Tm]) and to lincosamides (lincomycin [Ln]) was investigated in Mycoplasma synoviae, a pathogen that causes respiratory infections and synovitis in chicken and turkey. Sequence analysis of domains II and V of the two 23S rRNA alleles and ribosomal proteins L4 and L22 was performed on 49 M. synoviae isolates, M. synoviae type strain WVU1853, and reference strain FMT showing minimal inhibitory concentrations (MICs) to Ty (≤ 0.015 to 2 μg/ml), Tm (0.03 to ≥ 8 μg/ml), and Ln (0.125 to 8 μg/ml); MICs to Ery ranged from 32 to ≥ 128 μg/ml. Our results showed that the nucleotide substitution G748A (Escherichia coli numbering) in domain II of one or both 23S rRNA alleles may account for a slight increase in MICs to Ty and Tm (up to 0.5 and 2 μg/ml, respectively). No correlation between the presence of G748A and decreased susceptibility to Ln was found. However, the presence of the point mutations A2058G or A2059G in domain V of one or both alleles of the 23S rRNAs was correlated with a more significant decrease in susceptibility to Ty (1-2 μg/ml), Tm (≥ 8 μg/ml), and Ln (≥ 8 μg/ml). All M. synoviae isolates tested had a G2057A transition in the 23S rRNAs consistent with previously described intrinsic resistance to Ery. Mutations G64E (one isolate) and Q90K/H (two isolates) were identified in the L4 and L22 proteins, respectively, but their impact on decreased susceptibility to macrolides and lincomycin was not clear.

Multiple-locus VNTR Analyses of Methicillin-resistant Staphylococcus aureus from Jamaica.

BACKGROUNDThis study assessed the antimicrobial susceptibilities and the presence of inducible macrolide–lincosamide–streptogramin B (iMLSB) resistance in methicillin-resistant Staphylococcus aureus (MRSA) of Jamaica as well as the relatedness using polymerase chain reaction-based staphylococcal cassette chromosome mec (SCCmec) and multiple-locus variable numbers of tandem repeat analyses (MLVAs).MATERIALS AND METHODSAntimicrobial susceptibility, the presence of MLSB resistance, and SCCmec and MLVA patterns were assessed for 61 nonduplicate isolates of MRSA from hospitalized patients.RESULTSWhile no isolate was resistant to vancomycin, 53 (86.9%) isolates were resistant to ciprofloxacin, 52 (85.3%) to erythromycin, 49 (80%) to lincomycin, and 45 (74%) to clindamycin. Of the 52 erythromycin-resistant isolates, 48% exhibited constitutive resistance and 8% showed inducible MLSB (iMLSB) resistance. Most (85%) of typable isolates were SCCmec type IV, and among these, 16 MLVA patterns were identified.CONCLUSIONMultidrug resistance continues to characterize MRSA. Among the erythromycin-resistant isolates, constitutive resistance and iMLSB resistance are common. These facts will complicate the treatment of MRSA infections and warrant continued surveillance and judicial use of antimicrobial agents.

Macrolide- and tetracycline-resistance determinants of colonizing group B streptococcus in women in Egypt.

Group B streptococcus (GBS) is a commensal bacterium of the human gastrointestinal and genital tracts. It is a leading cause of neonatal sepsis and meningitis, and has also been recognized as an important pathogen in pregnant women and the elderly. We investigated mechanisms of macrolide and tetracycline resistance in GBS colonizing women in Egypt. A total of 100 isolates were screened using standard antibiotic susceptibility tests. A multiplex PCR assay was used to detect macrolide- and tetracycline-resistance determinants. All isolates were uniformly susceptible to penicillin G, ampicillin, cefotaxime, vancomycin and levofloxacin. The resistance rates to erythromycin, clindamycin, azithromycin, tetracycline and chloramphenicol were 17, 14, 16, 98 and 1 %, respectively. Among the erythromycin-resistant isolates, 82.4 % had constitutive macrolide-lincosamide-streptogramin B (cMLSB) resistance, 5.9 % had inducible MLSB (iMLSB) resistance and 11.8 % had M phenotype resistance. Among the cMLSB phenotypes, 64.3 % of isolates harboured the ermB gene and 35.7 % of isolates harboured none of the investigated macrolide-resistance genes. The single strain expressing the iMLSB phenotype possessed the ermA gene. Of the two strains with the M phenotype, only one possessed the mefA/E gene. Conversely, seven macrolide-sensitive strains (MIC <0.03 µg ml(-1)) were ermB positive and one macrolide-sensitive strain (MIC <0.03 µg ml(-1)) harboured mefA/E. Tetracycline resistance was predominantly due to tetM, which was detected alone (83.7 %) or in association with tetL (12.2 %), tetK (1 %) or tetO (1 %). One strain carried tetM associated with both tetL and tetK, and another strain carried tetO alone. The tetO strains were positive for the mefA/E gene, and the tetL and tetK carrier strains harboured the ermB gene. Susceptible strains harbouring but not expressing an antibiotic-resistance gene should be regarded as potentially resistant. These results emphasize the need to monitor the epidemiology of GBS antibiotic resistance in Egypt.

Molecular characterization of Streptococcus pyogenes group A isolates from a tertiary hospital in Lebanon.

Streptococcus pyogenes [Group A Streptococcus (GAS)] is one of the most important human pathogens, responsible for numerous diseases with diverse clinical manifestations. As the epidemiology of GAS infections evolves, a rapid and reliable characterization of the isolates remains essential for epidemiological analysis and infection control. This study investigated the epidemiological patterns and genetic characteristics of 150 GAS isolates from a tertiary hospital in Lebanon by emm typing, superantigens (SAgs) detection, PFGE and antibiotic profiling. The results revealed 41 distinct emm types, the most prevalent of which were emm89 (16 %), emm12 (10 %), emm2 (9 %) and emm1 (8 %). Testing for the presence of superantigens showed that speB (87 %), ssa (36 %) and speG (30 %) were predominant. PFGE detected 39 pulsotypes when a similarity cut-off value of 80 % was implemented. Antibiotic-susceptibility testing against seven different classes of antibiotics showed that 9 % of the isolates were resistant to clindamycin, 23 % were resistant to erythromycin and 4 % showed the macrolide-lincosamide-streptogramin B (MLSB) phenotype. The emergence of tetracycline-resistant strains (37 %) was high when compared with previous reports from Lebanon. This study provided comprehensive evidence of the epidemiology of GAS in Lebanon, highlighting the association between emm types and toxin genes, and providing valuable information about the origin and dissemination of this pathogen.

Clindamycin Resistance among Staphylococcus Aureus Isolated at Mbarara Regional Referral Hospital, in South Western Uganda.

AimsThe study was conducted to determine the prevalence of Clindamycin (CL) resistance and antimicrobial susceptibility among clinical isolates of Staphylococcus aureus (S. aureus) from Mbarara Regional Referral Hospital (MRRH) in Southwestern Uganda.Study DesignLaboratory based cross sectional study.Place and Duration of the StudyThe study was conducted at the Microbiology department of Mbarara Regional referral hospital between November 2012 and December 2013.MethodologyIn our study, we recruited 300 S. aureus isolates that were stored in the laboratory and were obtained from different clinical samples. The isolates were tested for antimicrobial susceptibility by phenotypic methods and for the genotypic expression of Macrolide Lincosamide StreptograminB (MLSB) resistance genes (ermA, ermB, ermC, and msrA). The D-test was also performed.ResultsPhenotypically, a total of 109 (36%) S. aureus isolates were resistant to CL, of which 9 (3%) were constitutively resistant while 100 (33.3%) were inducibly resistant. Genotypicaly, 134/300 (44.7%) isolates possessed at least one of the MLSB resistance genes. 23/300 (7.7%) tested positive for ermB, 98/300 (32.7%) tested positive for the ermC and 43/300 (14.3%) tested positive for the msrA genes with none possessing the ermA gene. Isolates were highly resistant to Sulfamethoxazole/trimethoprim, Erythromycin and Oxacillin with moderate resistance to Vancomycin and Imipenem and least resistance to LinezolidConclusionS. aureus resistance to CL was high in this set up. There was also high resistance to Sulfamethoxazole/trimethoprim, Erythromycin and Oxacillin but low resistance to Linezolid.

Genetic environment of the multi-resistance gene cfr in methicillin-resistant coagulase-negative staphylococci from chickens, ducks, and pigs in China

The present study focussed on the analysis of the genetic environment of the multi-resistance gene cfr detected among 21, mostly methicillin-resistant, coagulase-negative Staphylococcus (CoNS) isolates obtained from chickens, ducks and pigs in China. It included sequencing of the regions up- and downstream of the cfr gene on various plasmid types in 13 isolates, such as pSS-02 and pSS-02-like (n=7), pSS-03-like (n=1), pJP1-like (n=3), pSS-04 (n=1) and pJP2 (n=1). This analysis revealed that insertion sequences (IS21-558, IS256, IS257, or IS1216E) and other resistance genes (aacA-aphD and aadD for aminoglycoside resistance, ble for bleomycin resistance, fosD for fosfomycin resistance, erm(B) and erm(C) for macrolide–lincosamide–streptogramin B resistance, or fexA for phenicol resistance) coexisted on the respective plasmids. In the chromosomal copies of cfr identified in eight S. lentus isolates, the cfr gene was found to be bracketed by insertion sequences, such as IS256 or ISEnfa5. Stability tests confirmed that all chromosomal cfr-containing regions could be looped out via IS-mediated recombination. The observations made in this study extend the rather rudimentary knowledge about the genetic environment of cfr in staphylococci from chickens and ducks and confirmed that insertion sequences play an important role in the dissemination of cfr, not only among different types of plasmids, but also for the integration in the chromosomal DNA.

Identification of the novel spectinomycin resistance gene spw in methicillin-resistant and methicillin-susceptible Staphylococcus aureus of human and animal origin

Sir, Spectinomycin resistance in staphylococci is mostly mediated by spectinomycin 9-O-adenyltransferase encoded by the spc gene. This gene is located together with the macrolide–lincosamide– streptogramin B resistance gene erm(A) on transposon Tn554. The analysis of methicillin-resistant Staphylococcus aureus (MRSA) CC398 from cases of bovine mastitis showed that all spectinomycin-resistant isolates carried the gene spc along with erm(A), whereas MRSA CC398 isolates from turkey meat and turkey meat products occasionally exhibited spectinomycin resistance, but lacked the spc gene. This observation pointed towards the existence of other as yet unknown spectinomycin resistance genes in staphylococci. Most recently, the analysis of MRSA ST398 and methicillin-susceptible S. aureus (MSSA) ST9 isolates of human origin from Spain, but also MRSA ST9 isolates from swine in China, revealed the presence of multiresistance gene clusters that were most likely of enterococcal origin. – 7 So far, two different types of these multiresistance gene clusters have been identified, both of which carry the resistance genes aadE (streptomycin resistance), lnu(B) (lincosamide resistance) and lsa(E) (pleuromutilin– lincosamide–streptogramin A resistance). A closer look at these clusters revealed the presence of a reading frame for a putative spectinomycin resistance gene between aadE and lsa(E). This reading frame is 810 bp in size, has an unusual start codon (TTG) and codes for an adenyltransferase protein of 269 amino acids. Database searches identified identical or closely related proteins in Enterococcus faecium (e.g. GenBank accession number EFF20873), Enterococcus faecalis (e.g. GenBank accession number EEU82264) and Lactobacillus johnsonii (e.g. GenBank accession number EGP12870) (Figure 1). The corresponding proteins were referred to as streptomycin 3′′-adenylyltransferase, spectinomycin 9-O-adenylyltransferase, putative toxin-antitoxin system or a hypothetical protein in the respective database entries. Moreover, three database entries (GenBank accession numbers AAL05551, AFM38045 and AFU35063) reported a protein that was 27 amino acids shorter. This was due to the annotation of a wrong start codon (ATG) upstream of which no ribosome binding site was found. Analysis of the corresponding nucleotide sequences (GenBank accession numbers AF408195, JQ861959 and JX560992, respectively) confirmed the presence of the larger correct reading frame of 810 bp. To determine whether or not this protein confers resistance to streptomycin or spectinomycin, we decided to clone the gene and express it in an S. aureus host. For this, the recently developed PCR5 assay (forward: 5′-TTGGATTGCAGCATTATTGG-3′, reverse: 5′-ATTTGGTCGAAGCCTT GTTG-3′; annealing temperature 568C) was used to generate an amplicon of 1985 bp from the cloned 17.5 kb segment of plasmid pV7037 (GenBank accession number JX560992) from the porcine MRSA ST9 isolate SA7037. This amplicon included the entire reading frame for the putative resistance gene and 239 bp in the upstream and 936 bp in the downstream parts. The amplicon was initially cloned into pCR-Blunt II-TOPO (Life Technologies GmbH, Darmstadt, Germany), and the recombinant vector was transformed into Escherichia coli recipient strain TOP10. The amplicon was then cut off from this vector by EcoRI digestion and inserted into the single EcoRI site of the E. coli–S. aureus shuttle vector pLI50. This recombinant shuttle vector was first introduced by electrotransformation into E. coli TOP10 and subsequently into S. aureus RN4220. Antimicrobial susceptibility testing was performed by broth macrodilution for spectinomycin and streptomycin according to CLSI document M31-A3 with S. aureus ATCC 29213 as the quality control strain. The MICs of streptomycin were 4 mg/L for S. aureus RN4220, S. aureus RN4220 carrying pLI50 and S. aureus RN4220 carrying the recombinant pLI50, suggesting that the cloned gene does not confer streptomycin resistance. In contrast, the MICs of spectinomycin were 32 mg/L for S. aureus RN4220 and S. aureus RN4220 carrying pLI50, but were distinctly higher at 4096 mg/L for S. aureus RN4220 carrying the recombinant pLI50 with the cloned gene. This observation confirmed that the gene in question was a spectinomycin resistance gene, which was then designated spw. Besides the occurrence of spw in E. faecium, E. faecalis and L. johnsonii, a gene was detected in Streptococcus suis that encoded a protein of 262 amino acids (GenBank accession number AER19630) that showed 93.9% amino acid identity to Spw. In contrast, the Spw protein exhibited only 64.7% identity to the Tn554-associated Spc protein (Figure 1). To see whether spw was present in other staphylococci, a PCR assay (forward: 5′-CGGCAGTAATGGGTGGTTTA-3′, reverse: 5′-CAGCCACC TCAGATTCCATT-3′; annealing temperature 548C) was developed