Detection of Antibiotic Resistance and Biofilm-Producing Ability of Staphylococcus Species in Clinical Isolates

Background: Staphylococci are responsible for life-threatening infections in hospitals and community. Their ability to produce multiple virulence factors and antibiotic resistance is an important reason of high mortality in staphylococcal infections. Biofilm production by these organisms makes it difficult to treat. Most of the treating antibiotics are failing and making it a matter of concern. Aims?This study aims to detect the increased antibiotic resistance in biofilm-producing Staphylococcus and to compare the performance of three potential methods of detection. Methods?A total of 81 isolates of staphylococci including coagulase negative staphylococci (CoNs), methicillin resistant S. aureus (MRSA), and methicillin sensitive S. aureus (MSSA) are included in this study. After the identification, an antibiotic sensitivity test was performed. Biofilm detection was done by three different methods: Congo red agar method, tube adherence method, and microtiter plate method. Result?Out of the 81 samples, 37 CoNs, 17 MRSA, and 27 MSSA were identified. Out of them we got 43 (53%) biofilm producers by Congo red agar method, 40 (49%) by tube adherence method, and 52 (64%) producers by tissue culture plate/microtiter plate method. Most of the biofilm producers showed multiple drug resistance. Conclusion?We found out that the microtiter plate method is sensitive and reliable as compared with the other two methods. Antibiotic resistance was found to be very common in biofilm producers. This was due to the resistance developed as a result of the matrix that does not let the antibiotic bind with the organisms. This can make the treatment of Staphylococcus very difficult in the future as the rate of drug resistance is faster as compared with newly emerging antibiotics.

Multiresistance and virulence factors of Staphylococcus aureus isolated from pigs / [Multirresistência e fatores de virulência de Staphylococcus aureus isolados de suínos]

The emergence of livestock-associated methicillin-resistant Staphylococcus aureus strains (LA-MRSA) and the potential role of pigs in the evolution of these strains has led to increased interest in research of these microorganisms. However, this has contributed to a lack of research in the isolation and characterization of methicillin-susceptible S. aureus strains (MSSA). In this study, the prevalence of S. aureus in pigs in the nursery and finishing stages were analyzed. The susceptibility profiles to antibiotics, tolerance to heavy metals, and biofilm production of the isolates were evaluated using phenotypic and genotypic techniques. A total of 1,250 colonies suggestive of Staphylococcus spp. were isolated from 128 pigs, of which 63.6% (n = 795) belonged to this microbial genus. Sixty-seven colonies isolated from 34 animals (26.5%) were confirmed as S. aureus (8.4%). No strains resistant to copper, zinc, or methicillin were detected; however, all strains presented a resistance profile to at least three different classes of antimicrobials and 21 produced biofilms. These data are of concern, as they indicate the need for increased surveillance in the use of antimicrobials as well as reinforce the importance of studies on MSSA strains.(AU)

A emergência de cepas de Staphylococcus aureus resistentes à meticilina associadas à pecuária (LA-MRSA) e o papel potencial dos suínos na evolução dessas cepas têm levado ao aumento do interesse na pesquisa desses microrganismos. No entanto, isso tem contribuído para a falta de estudos sobre o isolamento e a caracterização de cepas de S. aureus sensíveis à meticilina (MSSA). Neste estudo, foi analisada a prevalência de S. aureus em suínos nas fases de creche e terminação. Os perfis de suscetibilidade aos antibióticos, a tolerância a metais pesados e a produção de biofilme dos isolados foram avaliados por meio de técnicas fenotípicas e genotípicas. Um total de 1.250 colônias sugestivas de Staphylococcus spp. foi isolado de 128 suínos, das quais 63,6% (n = 795) pertenciam a esse gênero microbiano. Sessenta e sete colônias isoladas de 34 animais (26,5%) foram confirmadas como S. aureus (8,4%). Nenhuma cepa resistente ao cobre, ao zinco ou à meticilina foi detectada; entretanto, todas as cepas apresentaram perfil de resistência a pelo menos três classes diferentes de antimicrobianos e 21 produziam biofilme. Esses dados são preocupantes, pois indicam a necessidade de maior vigilância no uso de antimicrobianos, bem como reforçam a importância de estudos com cepas de MSSA.(AU)

Virulence genes profile and biofilm formation of Methicillin-resistant and Methicillin-susceptible Staphylococcus aureus isolates from diabetic foot infections

Aims@#Diabetic foot infections (DFIs) represent one of the most important risk factors for lower extremity amputation. One of the major infection agents that causes DFIs is Staphylococcus aureus. Staphylococcus aureus is an important human pathogen causing variety of clinical manifestations which can lead to invasive infections, sepsis and even death. Outcomes of antibiotic treatment of diabetic foot infections may depend not only on the antimicrobial susceptibility of the etiological agents, but also their ability to produce diverse virulence factors. This study was aimed to investigate biofilm production and the presence of various virulence genes among Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-susceptible Staphylococcus aureus (MSSA) isolates obtained from patients with DFIs. @*Methodology and results@#A total of 48 clinical MRSA and MSSA isolates obtained from diabetic foot patients were studied for their biofilm formation and the presence of 29 known virulence genes. The biofilm formation was observed, analyzed and quantified using the microtiter plate method. Biofilm production was observed as 95.50% and 92.00% in the MRSA and MSSA isolates, respectively. Among the 29 virulence genes tested on the 48 clinical isolates, 19 virulence genes were detected. It was found that aap (62.50%), etd (54.17%), icaD (50.00%), aae (50.00%), seh (31.25%) and icaADB (22.92%) were the most prevalent genes. A total of 10 virulence genes (etb, gehD, icaB, icaC, seb, hla_haem, hld_epid, altE, fbe and sesI) were absent in all the isolates used.@*Conclusion, significance and impact of study@#Virulence genes play important role in clinical infections. Our results showed the presence rates of biofilm formation and accumulation-associated factors that are high among MRSA as well as MSSA isolates from DFIs. These results confirmed the importance of biofilm formation as regarded for DFIs.

Virulence, agr groups, antimicrobial resistance and epidemiology of Staphylococcus aureus isolated from bovine subclinical mastitis / Virulência, grupos agr, resistência a antimicrobianos e epidemiologia de isolados de Staphylococcus aureus obtidos de vacas com mastite

Fifty-two Staphylococcus aureus recovered from papillary ostium and milk samples collected from cows with subclinical mastitis and milking environments in three small dairy herds located in southeastern Brazil were subjected to PCR identification based on the thermonuclease (nuc) gene. All the strains were submitted to in vitro antimicrobial susceptibility testing, and we investigated the sequence types (STs), agr groups (I-IV), virulence genes encoding for Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs), biofilm-associated proteins, bi-component toxins, pyrogenic toxin superantigens, and enterotoxins. Screening for oxacillin resistance (2-6 µg/ml oxacillin), beta-lactamase activity assays, and PCR for the mecA/mecC genes detected 26 methicillin-susceptible S. aureus(MSSA) and 26 mec-independent oxacillin-nonsusceptible S. aureus (MIONSA). While MSSA isolates were found to be susceptible to all antimicrobial agents tested, or only resistant to penicillin and ampicillin, MIONSA isolates were multidrug-resistant. ST126-agr group II MSSA isolates were prevalent in milk (n=14) and carried a broad set of virulence genes (clfA, clfB, eno, fnbA, fiB, icaA, icaD, lukED, hla, and hlb), as well as the ST126-agr group II MIONSA isolated from milking liners (n=1), which also carried the eta gene. ST1-agr group III MIONSA isolates (n=4) were found in papillary ostium and milk, but most MIONSA isolates (n=21), which were identified in both papillary ostium and milking liners, were agr-negative and assigned to ST126. The agr-negative and agr group III lineages showed a low potential for virulence. Studies on the characterization of bovine-associated MSSA/MIONSA are essential to reduce S. aureus mastitis to prevent economic losses in dairy production and also to monitor the zoonotic potential of these pathogens associated with invasive infections and treatment failures in healthcare.

Cinquenta e dois isolados de Staphylococcus aureus obtidos de amostras colhidas do óstio papilar, do leite de vacas com mastite subclínica e do ambiente de ordenha em três fazendas de rebanhos leiteiros localizadas no sudeste do Brasil foram identificados por PCR para o gene da termonuclease (nuc). Todos os isolados foram testados para sensibilidade a antimicrobianos e foram investigados os sequence types (STs), grupos agr (I-IV) e genes de virulência que codificam Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs), proteínas associadas a biofilme, toxinas bi-componentes, toxinas pirogênicas com propriedades de superantígenos e enterotoxinas. Triagem para detecção de resistência à oxacilina (2-6 µg/ml oxacilina), ensaios de atividade de enzimas beta-lactamases e PCR para os genes mecA/mecC detectaram 26 estirpes de S. aureus sensíveis à meticilina (methicillin-susceptible S. aureus, MSSA) e 26 estirpes de S. aureus mec-negativas não sensíveis à meticilina (mec-independent oxacillin-nonsusceptible S. aureus, MIONSA). Enquanto os isolados MSSA foram sensíveis a todos os agentes antimicrobianos testados, ou apenas resistentes à penicilina e ampicilina, os isolados MIONSA foram multirresistentes. MSSA ST126-agr grupo II foram prevalentes no leite (n= 14) e apresentaram um amplo conjunto de genes de virulência (clfA, clfB, eno, fnbA, fiB, icaA, icaD, lukED, hla e hlb), assim como o isolado MIONSA ST126-agr grupo II proveniente de um insuflador (n= 1), o qual também apresentou o gene eta. MIONSA ST1-agr grupo III (n= 4) foram identificados no óstio papilar e leite, mas a maioria dos isolados MIONSA (n= 21), encontrados em óstios papilares e insufladores, foram agr-negativos e pertenceram ao ST126. As linhagens agr-negativas e agr grupo III apresentaram baixo potencial de virulência. Estudos sobre a caracterização de MSSA/MIONSA associados a bovinos são essenciais para a redução da mastite causada por S. aureus e de perdas econômicas na produção leiteira e, também, para o monitoramento do potencial zoonótico desses patógenos associados a infecções invasivas e falhas de tratamento em ambientes hospitalares.

Prevalence and Antimicrobial Susceptibility of Methicillin Resistant Staphylococcus in a Tertiary Care Hospital in Koshi Region (Northern Bihar)

Introduction: Nosocomial infection is a major problem globally. Methicillin-resistant Staphylococcus aureus (MRSA) remains one of the most important causes of nosocomial infections worldwide. MRSA are the important agents causing nosocomial infections. The study was conducted in the Department of Microbiology to determine the prevalence of MRSA and antibiotic susceptibility pattern. Material and Methods: This was a retrospective study conducted from July 2016 to August 2017 in a tertiary care hospital in Northern Bihar India. All isolates were identified in patients and data provided by the Clinical and Laboratory Standards Institute (CLSI) guidelines and antibiotic susceptibility pattern considered by Kirby Bauer disc diffusion method. The information was definitely recorded and analyzed using Microsoft Excel 2007 edition. Results: 200 Staphylococcus strains isolate of were isolated from various clinical samples, Out of 200 S. aureus isolates, 73 (36.5%) were methicillin resistant S. aureus (MRSA) and 127 (63.5%) were methicillin-sensitive S. aureus (MSSA) in our labs. Although, the majority of the MRSA isolates were resulted from pus samples 30, however, the S. aureus isolates resulted from post-operative wound infection was mostly MRSA. Conclusion: In conclusion, the importance of Isolation of MRSA patients and carriers in the hospitals, regular surveillance of hospital associated infections including monitoring antibiotic sensitivity pattern and strict drug policy for antibiotics may be helpful for reducing the incidence of these infections

High-level persistence of biofilm formation in in methicillin resistant Staphylococcus aureus

Aims@#Biofilm formation by Methicillin-resistant Staphylococcus aureus on a variety of surfaces and detection of the biofilm-forming population by the most reliable method is very much essential to diagnose the nosocomial infection caused by S. aureus. @*Methodology and results@#This study is aimed to evaluate the biofilm producing ability of S. aureus by qualitative Congo red agar (CRA), and quantitative microtitre plate (MTP) methods. The morphological difference of biofilms analysis was done by SEM (Scanning Electron Microscope) and genotyping analysis of mecA and femA for determination of MRSA among isolated S. aureus strains and to check the biofilm producers among MRSA strains. Biofilm production was found to be at different intensities by MTP. The strong, moderate and weak biofilm producers were found to be 38.63%, 31.81%, and 29.54% respectively. The strong adherent biofilm formed by representative isolate developed a dense biofilm with thick mucus three-dimensional multilayered structure of macroscopic dimension. Conversely, SEM analysis of moderate and weak biofilm representative strain failed to form a monolayer of scattered single cells to three-dimensional structure. The 47.72% of S. aureus isolates have shown positive for the genotypic analysis of mecA and femA. The strong and moderate biofilm forming MRSA was found to be 38.63% and 9.09%, respectively. @*Conclusion, significance and impact of study@#The great challenge is associated with biofilm mediated infection caused S. aureus healthy and hospitalized individual hence the present study reinforces the need of precautionary measures to avoid the indiscriminate use of antibiotics in case of biofilm-forming MRSA.

Carriage prevalence and drug resistance profile of MRSA and MSSA strains isolated from newborns / 中华疾病控制杂志

Objective To determine the carriage prevalence and antibiotics resistance profile of methicillin-resistant staphylococcus aureus(MRSA) and methicillin-sensitive staphylococcus aureus(MSSA) strains isolated from newborns. Methods A cross-sectional study was conducted in two hospitals in Shenzhen from August to November 2015. Cotton swab samples were collected from 1834 newborns. Staphylococcus aureus was identified by general laboratory tests, and antimicrobial-susceptibility profiles was tested. Results In total, 3.27% (60/1 834) Staphylococcus aureus (SA) strains were isolated, including 0.82% (15/1 834) MRSA and 2.45% (45/1 834) MSSA. Most MRSA and MSSA strains were resistant to penicillin and erythromycin but less than 10% strains resistant to linezolid. Drugs resistance rates of MRSA to erythromycin, moxifloxacin and clindamycin were all higher than that of MSSA (all P<0.05). The multidrug-resistant risk of MRSA (80.00%) was 12.36 times higher than that of MSSA (24.44%). Conclusions Multidrug-resistant SA especially MRSA strains isolated from newborns had the higher prevalence, the drugs resistance rate of MRSA strains was more serious than that of MSSA.

Prevalence and characteristics of livestock-associated methicillin-susceptible Staphylococcus aureus in the pork production chain in Korea

The emergence and prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) in livestock animals have become a worldwide public health concern. While the prevalence and genetic profiles of MRSA strains in pigs and pork meat have been actively studied, livestock-associated MSSA strains have only been characterized in a few small-scale studies. In this investigation, we assessed the nationwide prevalence of MSSA in the Korean pig production chain, including pig farms, slaughterhouses, and retail markets. Among the 41 MSSA strains, the predominant clonal lineages were sequence type (ST) 398 (n = 15, 37%) and ST5 (n = 13, 32%). Although the overall prevalence of MSSA (2.58%) was low and mostly restricted to pig farms, ST398 MSSA strains showed higher level of multidrug resistance phenotype versus non-ST398 MSSA strains. In addition to the MDR phenotype, all of the ST398 MSSA strains exhibited resistance to tetracycline as they harbored the tet(K), tet(L), and/or tet(M) genes. However, ST398 MSSA strains did not exhibit increased resistance to zinc compared with the non-ST398 strains. This study is the first to provide evidence of ST398 MSSA emergence in livestock animals in Korea. Further studies are necessary to elucidate the potential of ST398 MSSA strains for human transmission. Our findings suggest that the MDR phenotype and high levels of tetracycline resistance may have played an important role in the emergence and prevalence of ST398 MSSA in pig farms in Korea.

High frequency of methicillin-susceptible and methicillin-resistant Staphylococcus aureus in children under 1 year old with skin and soft tissue infections / Alta frequência de S. aureus (MSSA-MRSA) em crianças com menos de um ano deidade com infecções de pele e do tecido mole

Abstract Objective: Staphylococcus aureus is responsible for a large number of infections in pediatric population; however, information about the behavior of such infections in this population is limited. The aim of the study was to describe the clinical, epidemiological, and molecular characteristics of infections caused by methicillin-susceptible and resistant S. aureus (MSSA-MRSA) in a pediatric population. Method: A cross-sectional descriptive study in patients from birth to 14 years of age from three high-complexity institutions was conducted (2008-2010). All patients infected with methicillin-resistant S. aureus and a representative sample of patients infected with methicillin-susceptible S. aureus were included. Clinical and epidemiological information was obtained from medical records and molecular characterization included spa typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). In addition, staphylococcal cassette chromosome mec (SCCmec) and virulence factor genes were detected. Results: A total of 182 patients, 65 with methicillin-susceptible S. aureus infections and 117 with methicillin-resistant S. aureus infections, were included in the study; 41.4% of the patients being under 1 year. The most frequent infections were of the skin and soft tissues. Backgrounds such as having stayed in day care centers and previous use of antibiotics were more common in patients with methicillin-resistant S. aureus infections (p ≤ 0.05). Sixteen clonal complexes were identified and methicillin-susceptible S. aureus strains were more diverse. The most common cassette was staphylococcal cassette chromosomemec IVc (70.8%), which was linked to Panton-Valentine leukocidin (pvl). Conclusions: In contrast with other locations, a prevalence of infections in children under 1 year of age in the city could be observed; this emphasizes the importance of epidemiological knowledge at the local level.

Resumo Objetivo: O Staphylococcus aureus é responsável por um grande número de infecções na população pediátrica; contudo, as informações sobre o comportamento dessas infecções nessa população são limitadas. O objetivo do estudo foi descrever as características clínicas, epidemiológicas e moleculares de infecções causadas por Staphylococcus aureus suscetíveis e resistentes à meticilina (MSSA-MSRA) em uma população pediátrica. Método: Um estudo transversal descritivo foi realizado em pacientes entre 0 e 14 anos de idade de três instituições de alta complexidade (2008-2010). Todos os pacientes infectados com S. aureus resistentes à meticilina e uma amostra representativa de pacientes infectados com S. aureus suscetíveis à meticilina foram incluídos. As informações clínicas e epidemiológicas foram obtidas de prontuários médicos, e a caracterização molecular incluiu tipagem spa, Eletroforese em Gel de Campo Pulsado (PFGE) e Tipagem de sequências multilocus (MLST). Além disso, o Cassete Cromossômico Estafilocócico mec (SCCmec) e genes de fatores de virulência foram detectados. Resultados: 182 pacientes, 65 com infecções por S. aureus suscetíveis à meticilina e 117 com infecções por S. aureus resistentes à meticilina, foram incluídos no estudo; 41,4% dos pacientes com menos de um ano de idade. As infecções mais frequentes foram da pele e dos tecidos moles. Os históricos como internações em centros de atendimento e o uso prévio de antibióticos foram mais comuns em pacientes com infecções por S. aureus resistentes à meticilina (p ≤ 0,05). Dezesseis complexos clonais foram identificados, e as cepas de S. aureus suscetíveis à meticilina foram mais diversificadas. O cassete mais comum foi o Cassete Cromossômico Estafilocócicomec IVc (70,8%), relacionado à leucocidina de panton-valentine (pvl). Conclusões: Em comparação a outros locais, observamos uma prevalência de infecções em crianças com menos de um ano de idade na cidade; o que enfatiza a importância de conhecer a epidemiologia em nível local.

A Prospective Study on Bacterial Isolation from Diabetic Foot Ulcer in Department of Surgery

Background: There are various wound classification systems to assess the severity of diabetic foot ulcer and involve different factors specifically depth, site, presence of neuropathy, ischemia etc. Wagner’s system classification is the most widely used in grading of diabetic foot ulcers. Methods: The case study was carried out in the Department of Surgery, Geetanjali Medical College and Hospital, Udaipur. Results: 173 cases were enrolled in study and grade-3 Wagner’s ulcers were predominant. Males were predominant. Peripheral arterial disease was more common than peripheral neuropathy and both were found significantly associated with development of ulcers. Methicillin resistant Staphylococcus aureus and Klebsiella pneumoniae were most common isolates. MRSA exhibited maximum sensitivity to vancomycin, clindamycin and linezolid. Gram negative isolates exhibited maximum sensitivity to meropenems, piperacillin-tazobactam. Conclusions: This study conclude that prospective multicenter studies are required to assess the appropriate antibiotic regimen in diabetic foot ulcers and proper management of antibiotics must be started to decrease the incidence and development of MDR organisms.

Antibacterial steroidal alkaloids from Holarrhena antidysenteriaca / 中国天然药物

Two new steroidal alkaloids, isoconkuressine and N-formylconessimine, together with 6 known steroidal alkaloids including conkuressine, conessine, isoconessimine, conimine, conarrhimine, and funtudienine, were isolated from the seeds of Holarrhena antidysenteriaca Wall.ex A.DC. Their intrinsic antibacterial activities and synergistic effects with penicillin and vancomycin were analyzed in methicillin sensitive staphylococcus aureus (MSSA) and methicillin resistant staphylococcus aureus (MRSA). Two of the steroidal alkaloids including one new compound (N-formylconessimine) showed potential antibacterial activity and possessed synergistic effects with penicillin and vancomycin, respectively.

Local circulating clones of Staphylococcus aureus in Ecuador

ABSTRACT The spread of pandemic Staphylococcus aureus clones, mainly methicillin-resistant S. aureus (MRSA), must be kept under surveillance to assemble an accurate, local epidemiological analysis. In Ecuador, the prevalence of the USA300 Latin American variant clone (USA300-LV) is well known; however, there is little information about other circulating clones. The aim of this work was to identify the sequence types (ST) using a Multiple-Locus Variable number tandem repeat Analysis 14-locus genotyping approach. We analyzed 132 S. aureus strains that were recovered from 2005 to 2013 and isolated in several clinical settings in Quito, Ecuador. MRSA isolates composed 46.97% (62/132) of the study population. Within MRSA, 37 isolates were related to the USA300-LV clone (ST8-MRSA-IV, Panton-Valentine Leukocidin [PVL] +) and 10 were related to the Brazilian clone (ST239-MRSA-III, PVL−). Additionally, two isolates (ST5-MRSA-II, PVL−) were related to the New York/Japan clone. One isolate was related to the Pediatric clone (ST5-MRSA-IV, PVL−), one isolate (ST45-MRSA-II, PVL−) was related to the USA600 clone, and one (ST22-MRSA-IV, PVL−) was related to the epidemic UK-EMRSA-15 clone. Moreover, the most prevalent MSSA sequence types were ST8 (11 isolates), ST45 (8 isolates), ST30 (8 isolates), ST5 (7 isolates) and ST22 (6 isolates). Additionally, we found one isolate that was related to the livestock associated S. aureus clone ST398. We conclude that in addition to the high prevalence of clone LV-ST8-MRSA-IV, other epidemic clones are circulating in Quito, such as the Brazilian, Pediatric and New York/Japan clones. The USA600 and UK-EMRSA-15 clones, which were not previously described in Ecuador, were also found. Moreover, we found evidence of the presence of the livestock associated clone ST398 in a hospital environment.

Molecular characterization of community- & hospital-acquired methicillin-resistant & methicillin-sensitive Staphylococcus aureus isolates in Sikkim.

Background & objectives: The two major genotypic markers that distinguish community acquired (CA) from hospital acquired (HA) methicillin resistant staphylococcus aureus (MRSA) isolates are the architecture of mobile genetic element (SCCmec type) and presence of panton valentine leukocidin (PVL) toxin. This study was conducted to determine the molecular characteristics of CA- and HA- MRSA and methicillin sensitive S. aureus (MSSA) isolates in Sikkim. Methods: A total of 150 clinical isolates of S. aureus isolated from various clinical specimens were subjected to duplex (mec-A and pvl gene) and multiplex (SCCmec typing) PCR. Results: of the 150 isolates, 53 (35.33%) and 66 (44%) were positive for mec-A (MRSA) and pvl genes, respectively. Thirty eight (25.33%) met the definition of CA-MRSA and 15 (10%) of HA-MRSA and the remaining 63 (42%) and 34 (22.66%) as CA- and HA-MSSA, respectively. No significant difference was seen in the distribution of PVL toxin in MRSA and MSSA isolates, but it was significantly (P<0.001) high in overall MRSA isolates than in MSSA. The majority of the MRSA isolates showed a double amplification band of SCCmec type III plus V (54.71%), and only a fewer isolates were amplified by single DNA fragments of type I (1.88%), III (3.77%), IVa (1.88%) and V (11.32%). SCCmec types I, III, IVa, were found only in HA-MRSA isolates, whereas type V in both the CA- and HA-MRSA. AST pattern showed that 18.42 per cent (7/38) and 46.66 per cent (7/15) were multidrug resistant (MDR)-CA-MRSA and MDR-HA-MRSA, respectively. Interpretation & conclusions: The present results show that SCCmec type V MRSA has been on the rise, and genotypic markers such as pvl gene detection used for the differentiation of these clinically distinct isolates of MRSA may not be reliable.

Inducible clindamycin resistance in Staphylococcus aureus: Reason for treatment failure.

Emergence of methicillin resistance in Staphylococcus aureus (S.aureus) has left us with very few therapeutic alternatives available to treat staphylococcal infection. The widespread use of macrolide-lincosamide-streptogramin B (MLSB) antibiotics has led to an increase in number of staphylococcal strains acquiring resistance to MLSB antibiotics. This study was done to investigate the infections by hospital and community acquired “erythromycin- induced clindamycin resistant” strains or D-test positives of clinical isolates of Staphylococcus aureus (S.aureus) in a hospital. Three hundred isolates of S.aureus were subjected to routine antibiotic susceptibility testing including Cefoxitin (30µg) by modified disc diffusion method. Inducible resistance to clindamycin in S.aureus was tested by D-test as per CLSI guidelines. Among 300 S.aureus isolates, 114 (38%) were methicillin resistant Staphylococcus aureus (MRSA) and 186 (62%) methicillin susceptible Staphylococcus aureus (MSSA). Forty one (13.67%) isolates showed induced clindamycin resistance, 49(16.33%) showed constitutive resistance and 94 (31.33%) showed the MS phenotype. Inducible resistance and constitutive resistance were found to be higher in MRSA compared to MSSA (22.81%, 23.68% and 8.1%, 11.8% respectively). D-test should be included as a part of routine antibiotic susceptibility testing to detect induced clindamycin resistance to prevent treatment failure.

Rapid identification of methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylo-coccus aureus strains by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry / 中华微生物学和免疫学杂志

Objective To evaluate the capability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry ( MALDI-TOF MS) for rapid identification of methicillin-resistant Staphylo-coccus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) strains.Methods Twenty-five MRSA and thirty MSSA isolates were collected from the Second Affiliated Hospital of Zhejiang University School of Medicine as the experiment group .Twelve MRSA and twenty-two MSSA clinical strains were isola-ted as the control .All strains were identified by MALDI-TOF MS and the results were further analyzed by ClinProTools 3.0 software.Results Four algorithms including support vector machine (SVM), genetic al-gorithm ( GA) , supervised neural network ( SNN) and quick classifier ( QC) showed similar results in dis-tinguishing MRSA from MSSA isolates .The sensitivity of GA was 100 .0%and the sensitivities of other algo-rithms were all greater than 95.0%.The specificities of GA, SVM and QC were all greater than 90.0%. The areas under the receiver operating characteristic ( ROC) curves of four characteristic peaks at mass-to-charge ratios (m/z) of 3279, 6485, 6555 and 3299 m/z were all greater than 0.9.The virtual gel view showed that the bands generated by MSSA isolates at 3279 , 6485 and 6555 m/z were obviously deeper in color than those generated by MRSA isolates .However , the bands of MSSA isolates at 3299 m/z were appar-ently lighter in color than those of MRSA isolates .83.3%of MRSA and 90.0%of MSSA isolates from the control group were correctly identified by the GA model .Conclusion MALDI-TOF MS could rapidly and accurately identify MRSA from MSSA isolates under the strictly controlled experimental conditions with the advantages of less time-consuming, high sensitivity and high specificity .The accurate identification of MRSA from MSSA isolates could be applied for the prevention and treatment of MRSA infection .

Study of the virulence genes and molecular characteristics of Staphylococcus aureus strains / 中华微生物学和免疫学杂志

Objective To investigate the molecular characteristics and virulence genes of 488 Staphylococcus aureus ( S.aureus) strains isolated from the People′s Hospital of Huangzhou District in Hubei Province during 2009 to 2013.Methods The minimum inhibitory concentrations (MICs) of Oxacillin and Cefoxitin to S.aureus were determined by agar dilution method .PCR analysis was used for the detection of staphylococcal cassette chromosome mec ( SCCmec ) and multilocus-sequence typing ( MLST ) .Multiplex PCR analysis was performed to detect the 31 common virulence genes .Results A total of 227 methicillin-resistant S.aureus ( MRSA) strains were identified from 488 S.aureus strains with a prevalence rate of 46.5%.The SCCmec Ⅲtype was the prevalent genotype accounting for 81.5% of the 227 MRSA strains, followed by Ⅳtype which accounted for 10.1%.The predominant clonal complex ( CC) of MRSA strains was CC8 accounting for 81.1%, followed by CC59 (4.8%) and CC5 (3.1%).CC1 was the predominant clonal complex of methicillin-sensitive S.aureus (MSSA) strains, accounting for 34.1% of the 261 MSSA strains, followed by CC398 (21.8%), CC121 (14.9%) and CC59 (13.0%).The number of MSSA iso-lates carrying no less than 15 test virulence genes was 109 ( 48 .2%) , which was significantly higher than that of MRSA isolates (28.2%) (P=0.002).A close relationship between the enterotoxins genes (sed, sej and ser) and the CCs of CC8 and CC5 was identified.Exfoliatin genes (eta and etb) and lukED gene were detected only in strains that belonged to CC 1.Strains that belonged to CC 1 and CC59 clones showed higher rates of pvl gene as compared with those belonging to other CCs (P<0.05).Conclusion The prevalence rate of MRSA strains was 46.5%in Huangzhou District, Hubei Province, which was consistent with the na-tional average rate .The predominant genotype of MRSA strains was ST 239-MRSA-SCCmecIII , accounting for 79.3%.Effective measures should be taken by Health sectors to control the spread of MRSA strains .The MSSA isolates carried more virulence genes than MRSA strains .The spectrums of virulence genes were var-ied in strains belong to different CCs clones , indicating the close relationship between virulence genes and genetic backgrounds .

Characterization of Staphylococcus aureus isolated from clinical specimens by matrix assisted laser desorption/ionization time-of-flight mass spectrometry / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To develop a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approach to identify Staphylococcus aureus (S. aureus) and differentiate methicillin-resistant S. aureus (MRSA) from methicillin-sensitive S. aureus (MSSA).</p><p><b>METHODS</b>A total of 100 S. aureus strains isolated from clinical specimens and farm workers were collected and analyzed by MALDI-TOF-MS. And data obtained were interpreted with biotyper software.</p><p><b>RESULTS</b>Ninety-two strains were identified by MALDI-TOF-MS as S. aureus at a level of secure genus and probable species, and 4 strains were identified at probable genus after their cultivation, spectral collection and data preprocessing. One strain was identified as S. aureus with lower score. It was revealed that identification of S. aureus by MALDI-TOF-MS was highly correlated with typing by biochemical and serological methods with an accuracy as high as 97%. The biotyper cluster analysis showed that 100 isolates were divided into 2 types at the distance level of 400. Higher peak intensity in the mass of both 3784 Da and 5700 Da was observed in MRSA, whereas that was absent from MSSA.</p><p><b>CONCLUSION</b>MALDI-TOF-MS is considered a simple, rapid and highly reproducible technique with high-throughput and accuracy for the identification of S. aureus and it can reliably differentiate MRSA from MSSA.</p>

A report on infection dynamics of inducible clindamycin resistance of Staphylococcus aureus isolated from a teaching hospital in India

<p><b>OBJECTIVE</b>To investigate the infection of hospital- and community-acquired "erythromycin-induced clindamycin resistant" strains or D-test positives of clinical isolates of Staphylococcus aureus (S. aureus) (with and without methicillin resistance) in a hospital.</p><p><b>METHODS</b>Strains of S. aureus isolated from clinical specimens were subjected to D-test and antibiotic profiling.</p><p><b>RESULTS</b>Of the total 278 isolates, 140 (50.35%) were D-test positives and the rest were D-test negatives. Further, of 140 (100%) positives, 87 (62.14%) and 53 (37.85%) strains were from males and females, respectively. Of 140 (100%) positives, 117 (83.57%) were methicillin resistant S. aureus and 23 (16.42%) were methicillin sensitive S. aureus; of 140 strains, 103 (73.57%) strains from persons with and 37 (26.42%) were without related infections; of 140 strains, 91 (65%) and 49 (35%) were from hospital- and community-acquired samples, respectively. In 140 strains, 118 (84.28%) with comorbidities and 22 (15.71%) without comorbidities cases were recorded; similarly, persons with prior antibiotic uses contributed 108 (77.14%) and without 32 (22.85%) positive strains. These binary data of surveillance were analyzed by a univariate analysis. It was evident that the prior antibiotic uses and comorbidities due to other ailments were the determinative factors in D-test positivity, corroborated by low P values, P=0.001 1 and 0.002 4, respectively. All isolates (278) were resistant to 17 antibiotics of nine groups, in varying degrees; the minimum of 28% resistance for vancomycin and the maximum of 97% resistance for gentamicin were recorded. Further, of 278 strains, only 42 (15.1%) strains were resistant constitutively to both antibiotics, erythromycin resistant and clindamycin resistant, while 45 (16.2%) strains were constitutively sensitive to both antibiotics (erythromycin sensitive and clindamycin sensitive). Further, of the rest 191 (68.7%) strains were with erythromycin resistant and clindamycin resistant, of which only 140 (50.35%) strains were D-test positives, while the rest 51 (18.34%) strains were D-test negatives.</p><p><b>CONCLUSIONS</b>In view of high prevalence of D-test positive S. aureus strains, and equally high prevalence of multidrug resistant strains both in community and hospital sectors, undertaking of D-test may be routinely conducted for suppurative infections.</p>

An epidemiologic investigation of chronic osteomyelitis among pediatric patients admitted from 2006 to 2010 at the Philippine General Hospital

Background@#Osteomyelitis is a debilitating disease if not properly treated. Epidemiologic and microbiologic data will be of great importance in the direction of treatment. @*Objective@#To determine the epidemiologic and clinical profile of pediatric patients with chronic osteomyelitis admitted at Philippine General Hospital from 2006 to 2010. @*Methodology@#This is a retrospective study involving a review of medical records of pediatric patients with chronic osteomyelitis admitted at the Philippine General Hospital during the 5 year study period. Frequencies and percentages were computed for nominal data. Comparison of the different variables was done using Chisquare and Fisher Exact test. @*Results@# Eighty of the 134 cases of pediatric patients with chronic osteomyelitis were reviewed. Twenty-three percent of all operations involved the femur (N=18) and tibia (N=18). On radiograph, the presence of sequestrum was the most common finding noted in 53% of the cases. The predominant organism isolated in bone cultures was MSSA (40%) followed by MRSA (20%). On tissue cultures MRSA was the most common isolate in 34%, followed by MSSA(31%). Majority of the patients were given Oxacillin as empiric therapy (55%).No significant difference was observed with respect to the areas of bone involvement, signs and symptoms, radiologic findings and laboratory parameters between MSSA and MRSA osteomyelitis (p>0.05). @*Conclusion@#There were 239 per 100,000 cases of chronic osteomyelitis. The most common bones involved were the femur and tibia with sequestrum as the most common radiologic finding. In the previous studies, MRSA was not reported but was noted in the present study. Most of the patients in our study were treated with both antibiotics and surgery to optimize management. Ninety-eight percent of the cases had significant clinical improvement upon discharge. Based on this study, laboratory parameters, clinical manifestations and area involved cannot be utilized in differentiating MRSA from MSSA osteomyelitis. Further studies are needed to support our findings and isolation of the organism is still required for definitive identification to distinguish between MRSA from MSSA osteomyelitis.

Characterisation of virulence genes in methicillin susceptible and resistant Staphylococcus aureus isolates from a paediatric population in a university hospital of Medellín, Colombia

Virulence and antibiotic resistance are significant determinants of the types of infections caused by Staphylococcus aureus and paediatric groups remain among the most commonly affected populations. The goal of this study was to characterise virulence genes of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains isolated from a paediatric population of a Colombian University Hospital during 2009. Sixty MSSA and MRSA isolates were obtained from paediatric patients between zero-14 years. We identified the genes encoding virulence factors, which included Panton-Valentine leucocidine (PVL), staphylococcal enterotoxins A-E, exfoliative toxins A and B and toxic shock syndrome toxin 1. Typing of the staphylococcal chromosome cassette mec (SCCmec) was performed in MRSA strains. The virulence genes were more diverse and frequent in MSSA than in MRSA isolates (83 percent vs. 73 percent). MRSA strains harboured SCCmec types IVc (60 percent), I (30 percent), IVa (7 percent) and V (3 percent). SCCmec type IVc isolates frequently carried the PVL encoding genes and harboured virulence determinants resembling susceptible strains while SCCmec type I isolates were often negative. PVL was not exclusive to skin and soft tissue infections. As previously suggested, these differences in the distribution of virulence factor genes may be due to the fitness cost associated with methicillin resistance.