Caracterización fenotípica y molecular de aislamientos clínicos de staphylococcus hominis, staphylococcus cohnii y staphylococcus sciuri.

Staphylococcus hominis, Staphylococcus cohnii y Staphylococcus sciuri se encuentran clasificados dentro del grupo de los Estafilococos Coagulasa Negativos (ECN). Los ECN están entre los principales agentes causales de enfermedades asociadas a la atención de la salud, principalmente bacteriemias relacionadas con el uso de catéter. El principal factor de virulencia descrito para los ECN es la capacidad de producir biopelícula, en donde su formación está asociada a la colonización de dispositivos médicos y a un incremento en la resistencia a los antibióticos. El objetivo de este trabajo fue caracterizar la formación de biopelícula, los genes asociados a esta producción, la resistencia a meticilina, el tipo de SCCmec, la relación genética de los aislamientos y la determinación del perfil de susceptibilidad a los antibióticos en células planctónicas y de biopelícula en aislamientos clínicos de S. hominis, S. cohnii y S. sciuri. El estudio incluyó a 67 aislados de S. hominis, 23 S. cohnii y 11 S. sciuri provenientes de especímenes clínicos. La resistencia a la meticilina se evaluó con la prueba de disco de cefoxitina. La detección del gen mecA y SCCmec se realizó por PCRs múltiples. La relación genética se determinó por electroforesis en gel de campos pulsados. La formación de biopelícula se evaluó por tinción con violeta cristal y se determinó el índice de biopelículas (IB). La susceptibilidad a los antibióticos de las células planctónicas (concentración mínima inhibitoria, CMI) y las células del biopelícula (concentración mínima de erradicación de biopelícula, CMEB) se determinaron por el método de dilución en caldo. Los resultados indicaron que más del 85% de los aislamientos fueron resistentes a la meticilina y presentaron el gen mecA positivo. De los aislamientos mecA positivos, más del 66% presentaron un tipo de SCCmec diferente al descrito para S. aureus. La clonalidad en general fue baja, pero se detectaron al menos tres clonas en cada especie, las cuales incluyeron de dos a ochos aislamientos. Más del 80% de los aislamientos se clasificaron como productores de biopelícula y el 91% de los aislamientos de S. hominis presentaron una fuerte producción de esta. Más del 60% de los aislamientos presentaron el gen icaD. Se observó que a mayor IB, mayor fue el valor de CMEB respecto al de CMI para amikacina, vancomicina, linezolid, oxacilina, ciprofloxacina y cloranfenicol. En conclusión, los aislamientos de Staphylococcus hominis, Staphylococcus cohnii y Staphylococcus sciuri presentaron una frecuencia alta de resistencia a meticilina y a otros antibióticos. La mayoría de los tipos de SCCmec detectados fueron diferentes a los descritos para S. aureus. Se encontró baja clonalidad. Los resultados indican que las tres especies son formadoras de biopelícula y S. hominis es un productor fuerte. La producción de biopelícula se asoció con un incremento en la resistencia a los antibióticos

Chlorhexidine whole-body washing of patients reduces methicillin-resistant Staphylococcus aureus and has a direct effect on the distribution of the ST5-MRSA-II (New York/Japan) clone

Abstract Purpose. Methicillin-resistant Staphylococcus aureus (MRSA) colonizes the skin of hospitalized patients and is associated with high morbidity and mortality. To prevent colonization and infection by S. aureus, better disinfection practices are required. Therefore, we evaluated the effect of chlorhexidine whole-body washing on hospital-acquired S. aureus infections among intensive care unit (ICU) patients in a tertiary hospital in Mexico. Methodology. The study was conducted over 18 months to evaluate the effect of 2% chlorhexidine gluconate (CXG) wholebody washing of ICU adult patients on chlorhexidine and antibiotic resistance, biofilm production and clonal distribution of S. aureus in a tertiary care hospital. Minimum inhibitory concentrations for CXG, antibiotic susceptibility and biofilm production by S. aureus isolates were determined. Pulsed-field gel electrophoresis, multilocus sequence typing (MLST) and PCR for Panton–Valentine leucocidin (PVL) were used for molecular typing of MRSA isolates. Results/Keyfindings. We included 158 isolates. A reduction in antibiotic resistance in the study period was observed for clindamycin, levofloxacin, norfloxacin, oxacillin and trimethoprim/sulfamethoxazole. None of the isolates showed reduced susceptibility to CXG. Most of the isolates were non-biofilm producers (147/158). The most commonly identified clone was a descendant of the ST5-MRSA-II (New York/Japan) clone. This clone decreased during the intervention period and reappeared markedly in the post-intervention period. During the post-intervention period, two isolates were related with the clone ST8-MRSA-IV (also known as USA300). Conclusion. Our findings suggest that the CXG bathing favored the reduction of healthcare-associated MRSA isolates and a temporary reduction of the predominant ST5-MRSA-II (New York/Japan) clone

Microbiological and Molecular Characterization of Staphylococcus hominis Isolates from Blood

Background: Among Coagulase-Negative Staphylococci (CoNS), Staphylococcus hominis represents the third most common organism recoverable from the blood of immunocompromised patients. The aim of this study was to characterize biofilm formation, antibiotic resistance, define the SCCmec (Staphylococcal Chromosomal Cassette mec) type, and genetic relatedness of clinical S. hominis isolates. Methodology: S. hominis blood isolates (n = 21) were screened for biofilm formation using crystal violet staining. Methicillin resistance was evaluated using the cefoxitin disk test and the mecA gene was detected by PCR. Antibiotic resistance was determined by the broth microdilution method. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) and SCCmec typed by multiplex PCR using two different methodologies described for Staphylococcus aureus. Results: Of the S. hominis isolates screened, 47.6% (10/21) were categorized as strong biofilm producers and 23.8% (5/21) as weak producers. Furthermore, 81% (17/21) of the isolates were methicillin resistant and mecA gene carriers. Resistance to ampicillin, erythromycin, and trimethoprim was observed in .70% of isolates screened. Each isolate showed a different PFGE macrorestriction pattern with similarity ranging between 0–95%. Among mecA-positive isolates, 14 (82%) harbored a non-typeable SCCmec type: eight isolates were not positive for any ccr complex; four contained the mec complex A ccrAB1 and ccrC, one isolate contained mec complex A, ccrAB4 and ccrC, and one isolate contained the mec complex A, ccrAB1, ccrAB4, and ccrC. Two isolates harbored the association: mec complex A and ccrAB1. Only one strain was typeable as SCCmec III. Conclusions: The S. hominis isolates analyzed were variable biofilm producers had a high prevalence of methicillin resistance and resistance to other antibiotics, and high genetic diversity. The results of this study strongly suggested that S. hominis isolates harbor new SCCmec structural elements and might be reservoirs of ccrC1 in addition to ccrAB1 and mec complex A

Antibiotic Susceptibility of Biofilm Cells and Molecular Characterisation of Staphylococcus hominis Isolates from Blood

Objectives We aimed to characterise the staphylococcal cassette chromosome mec (SCCmec) type, genetic relatedness, biofilm formation and composition, icaADBC genes detection, icaD expression, and antibiotic susceptibility of planktonic and biofilm cells of Staphylococcus hominis isolates from blood. Methods The study included 67 S. hominis blood isolates. Methicillin resistance was evaluated with the cefoxitin disk test. mecA gene and SCCmec were detected by multiplex PCR. Genetic relatedness was determined by pulsed-field gel electrophoresis. Biofilm formation and composition were evaluated by staining with crystal violet and by detachment assay, respectively; and the biofilm index (BI) was determined. Detection and expression of icaADB Cgenes were performed by multiplex PCR and real-time PCR, respectively. Antibiotic susceptibilities of planktonic cells (minimum inhibitory concentration, MIC) and biofilm cells (minimum biofilm eradication concentration, MBEC) were determined by the broth dilution method. Results Eighty-five percent (57/67) of isolates were methicillin resistant and mecA positive. Of the mecA-positive isolates, 66.7% (38/57) carried a new putative SCCmec type. Four clones were detected, with two to five isolates each. Among all isolates, 91% (61/67) were categorised as strong biofilm producers. Biofilm biomass composition was heterogeneous (polysaccharides, proteins and DNA). All isolates presented the icaD gene, and 6.66% (1/15) isolates expressed icaD. This isolate presented the five genes of ica operon. Higher BI and MBEC values than the MIC values were observed for amikacin, vancomycin, linezolid, oxacillin, ciprofloxacin, and chloramphenicol. Conclusions S. hominis isolates were highly resistant to methicillin and other antimicrobials. Most of the detected SCCmec types were different than those described for S. aureus. Isolates indicated low clonality. The results indicate that S. hominis is a strong biofilm producer with an extracellular matrix with similar composition of proteins, DNA and N-acetylglucosamine; and presents high frequency and low expression of icaD gene. Biofilm production is associated with increased antibiotic resistance

The successful containment of a hospital outbreak caused by NDM-1-producing Klebsiella pneumoniae ST307 using active surveillance

The worldwide dissemination of high-risk carbapenemase-producing Klebsiella pneumoniae clones has become a major threat to healthcare facilities. This study describes the successful containment of a hospital outbreak caused by NDM-1-producing K. pneumoniae Sequence Type (ST) 307 using active surveillance. The outbreak began when a patient was transferred from a local hospital. After 48 hours in our hospital, a tracheal aspirate was positive for a meropenem resistant and carbapenemase-producing K. pneumoniae. All patients in the medical intensive care unit (ICU) and the neurology wards were subject to contact precautions. The hospital surfaces and devices, healthcare workers, and patients from these wards were screened by cultures. Fecal swabs were placed into broth and PCR for blaKPC, blaOXA-48, blaIMP, blaVIM, and blaNDM, which were performed directly from the broth after 12 hours. PCRs were also performed on DNA extracted from carbapenemase-producing species from subcultured broths. Five and nine days later, two more patients’ rectal swabs tested positive. Molecular assays identified K. pneumoniae blaNDM-1 onto a 130-kb conjugative plasmid (IncY, IncFIIs, and IncFIIY), ST307. After the three patients were discharged, monitoring continued, and after three weeks with negative results, rectal swabbing ended. In conclusion, it was possible to contain a hospital outbreak caused by NDM-1-producing K. pneumoniae ST307 through epidemiological and microbiological surveillance. With the methodology used, the detection of NDM-type genes in fecal samples was obtained in approximately 15 hours after obtaining the fecal sample

The carriage of interleukin-1B-31*C allele plus Staphylococcus aureus and Haemophilus influenzae increases the risk of recurrent tonsillitis in a Mexican population

Abstract The aim of the present study was to estimate the relative contribution of immunogenetic and microbiological factors in the development of recurrent tonsillitis in a Mexican population. Patients (n = 138) with recurrent tonsillitis and an indication of tonsillectomy (mean age: 6.05 years±3.00; median age: 5 years, female: 58; age range: 1–15 years) and 195 nonrelated controls older than 18 years and a medical history free of recurrent tonsillitis were included. To evaluate the microbial contribution, tonsil swab samples from both groups and extracted tonsil samples from cases were cultured. Biofilm production of isolated bacteria was measured. To assess the immunogenetic component, DNA from peripheral blood was genotyped for the TNFA-308G/A single-nucleotide polymorphism (SNP) and for the IL1B -31C/T SNP. Normal microbiota, but no pathogens or potential pathogens, were identified from all control sample cultures. The most frequent pathogenic species detected in tonsils from cases were Staphylococcus aureus (48.6%, 67/138) and Haemophilus influenzae (31.9%, 44/138), which were found more frequently in patient samples than in samples from healthy volunteers (P<0.0001). Importantly, 41/54 (75.9%) S. aureus isolates were biofilm producers (18 weak and 23 strong), whereas 17/25 (68%) H. influenzae isolates were biofilm producers (10 weak, and 7 strong biofilm producers). Patients with at least one copy of the IL1B-31*C allele had a higher risk of recurrent tonsillitis (OR = 4.03; 95% CI = 1.27– 14.27; P = 0.013). TNFA-308 G/A alleles were not preferentially distributed among the groups. When considering the presence of IL1B-31*C plus S. aureus, IL1B-31*C plus S. aureus biofilm producer, IL1B-31*C plus H. influenzae or IL1B-31*C plus H. influenzae biofilm producer, the OR tended to infinite. Thus, the presence of IL1B-31*C allele plus the presence of S. aureus and/or H. influenzae could be related to the development of tonsillitis in this particular Mexican population

Risk factors and molecular mechanisms associated with trimethoprim–sulfamethoxazole resistance in Stenotrophomonas maltophilia in Mexico

Abstract Purpose. Stenotrophomonas maltophilia is a multidrug-resistant opportunistic pathogen causing an increasing number of nosocomial infections. Our aim was to evaluate the risk factors and mechanisms associated with trimethoprim– sulfamethoxazole (SXT) resistance in S. maltophilia infections in Mexico. Methodology. Clinical isolates and patients’ demographic and clinical data were collected from February 2007 to August 2015 in two tertiary-care hospitals in Mexico. Antimicrobial susceptibility and analysis of sul and SmeABC and SmeDEF efflux pump overexpression were performed in all isolates. Results/Key findings. In the 9-year period, 196 patients infected with S. maltophilia were identified. Most patients were male, and the mean age was 46.2years. The mean Charlson score was 1.42, and the most frequent comorbidities were arterial hypertension (26.7%), type 2 diabetes (21.2%) and cerebral infarction (11.6%). High drug resistance to meropenem (93.4%), gentamicin (55.1%), ceftazidime (52.3%), cefotaxime (51.5%), amikacin (42.3%) and cefepime (32.1%), and lower resistance to ciprofloxacin (26.0%), SXT (25.0%), chloramphenicol (14.3%) and levofloxacin (2.6%) were detected. SXT resistance was not associated with the sul genes. SmeABC overexpression was associated with gentamicin (P=0.001) and levofloxacin resistance (P=0.041), whereas SmeDEF overexpression was associated with ceftazidime resistance (P=0.003). Prolonged hospitalization (�15days) was an independent risk factor for SXT-resistant S. maltophilia infections (OR=3.05; 95%CI=1.12– 8.86; P=0.029). Conclusion. Given the high SXT resistance rate, SXT is not an effective first-line therapy for our patients; instead, levofloxacin could be used as an appropriate therapeutic option against S. maltophilia infections

Sexually transmitted pathogens, coinfections and risk factors in patients attending obstetrics and gynecology clinics in Jalisco, Mexico

Objetivo. Determinar la frecuencia de nueve patógenos de transmisión sexual, coinfecciones y factores de riesgo en pacientes que acudieron a una consulta de ginecología y obstetricia en Jalisco, México. Material y métodos. Se analizaron muestras de 662 pacientes que asistieron a la consulta de ginecología y obstetricia. Se detectaron Treponema pallidum, VIH y VHC mediante serología. Se detectó VPH por Reacción de Cadena de Polimerasa (PCR) y sus genotipos se detectaron por Polimorfismos de Longitud de Fragmentos de Restricción (RFLP). Se detectaron Trichomonas vaginalis, VHS-1, VHS-2, Mycoplasma genitalium, Neisseria gonorrhoeae y T. pallidum por PCR múltiple. Resultados. Por serología, la frecuencia de VIH fue 6.8%, de T. pallidum fue 2.26% y de VHC fue 0.15%. Por PCR, la frecuencia más alta fue de VPH (13.9%, el genotipo más frecuente fue el 16, 33.7%), seguida de T. vaginalis (14.2%), VHS-1 (8.5%), M. genitalium (2.41%), N. gonorrhoeae (2.11%), VHS-2 (1.8%) y T. pallidum (1.05%). Los pacientes infectados con T. vaginalis presentaron más probabilidades de tener múltiples coinfecciones (p = 0.01). Conclusiones. La frecuencia de infección por VPH, VHS-1, VHS-2, M. genitalium y T. vaginalis fue menor a lo reportado. Sin embargo, se detectó una alta frecuencia de VIH, T. pallidum, y N. gonorrhoeae. ABSTRACT Objective. To determine the frequency of nine sexually transmitted pathogens, coinfections and risk factors in patients attending obstetrics and gynecology clinics in Jalisco, Mexico. Materials and methods. Samples from 662 patients attending obstetrics and gynecology clinics were analyzed. Treponema pallidum, HIV, and HCV were detected by serology. HPV was detected by Polimerase Chain Reac- tion (PCR), and its genotype was determined by Restriction Fragment Length Polymorphism (RFLP). Trichomonas vaginalis, HSV-1, HSV-2, Mycoplasma genitalium, Neisseria gonorrhoeae and T. pallidum were detected by multiplex PCR. Results. By serology, HIV frequency was 6.8%, T. pallidum was 2.26%, and HCV was 0.15%. By PCR, HPV frequency was 13.9%, (more frequent genotype was 16, 33.7%), followed by T. vaginalis (14.2%), HSV-1 (8.5%), M. genitalium (2,41%), N. gonorrhoeae (2.11%), HSV-2 (1.8%), and T. pallidum (1.05%). Patients infected with T. vaginalis were more likely to have multiple coinfections (p = 0.01). Conclusion. The frequency of HPV, HVS-1, HSV-2, M. genitalium and T. vaginalis was lower than that reported. However, a high frequency of HIV, T. pallidum, and N. gonorrhoeae was detected