Genetic relatedness of clinical and environmental Acinetobacter baumanii isolates from an intensive care unit outbreak.

INTRODUCTION: Determination of microbial genetic relatedness is important for improving efficiency of infection control measures during hospital outbreaks. This study aimed to analyze the clonal relationships of clinical and environmental Acinetobacter baumannii strains isolated during an outbreak in the intensive care unit (ICU) of a secondary care hospital in Saudi Arabia. METHODOLOGY: Twelve clinical and fourteen environmental A. baumannii isolates identified during an outbreak in February 2013 in the 14-bed adult intensive care unit of a tertiary care hospital in Riyadh, Saudi Arabia, were studied. Bacterial identification and antimicrobial susceptibility testing were carried out using Microscan Walkaway 96 automated system. Determination of clonal diversity was investigated by repetitive-sequence-based polymerase chain reaction (rep-PCR) with the semi-automated DiversiLab system. RESULTS: The majority of the clinical isolates were from endotracheal tube aspirates (n = 9), one from a wound swab and two were from urine and sputum, respectively. Environmental isolates were from bed railings (n = 10) and with one each from curtain, stethoscope, computer mouse and telephone. Isolates were categorized into 6 clusters (Groups 1-6). Most of the isolates were associated with two clusters namely Groups 2 (n = 11) and 3 (n = 9). All isolates were multidrug resistant showing resistance to three or more classes of antibiotics. One clinical strain from Cluster 3 was resistant to colistin (MIC > 4ug/ml). CONCLUSION: This outbreak was caused by two clonal groups of multidrug resistant A. baumannii. The presence of multiple environmental clones was suggestive of environmental source dissemination via healthcare workers within the ICU.

Molecular characteristics of extended-spectrum ß-lactamase-producing Escherichia coli in Riyadh: emergence of CTX-M-15-producing E. coli ST131.

BACKGROUND: The prevalence of extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-EC) has increased recently. The aim of this study was to further characterise and to assess the occurrence of ESBL-EC in Riyadh, to use pulsed field gel electrophoresis (PFGE) typing to investigate the epidemiology of ESBL-EC and to determine the prevalence of ST131 in ESBL-EC. METHODS: A total of 152 E. coli isolates were collected at a tertiary hospital in Riyadh from September 2010 to June 2011. Genotypic and phenotypic methods were used to characterise ESBLs. PFGE was used to determine genetic relatedness. Detection of ST131 and CTX-M-like ESBLs was performed using real-time PCR. RESULTS: Of 152 strains, 31 were positive for ESBLs by phenotypic methods. The blaCTX-M-15 gene was highly prevalent (30/31 strains, 96.77%) among the 31 ESBL-positive E. coli strains. The blaCTX-M-27 gene was detected in one strain. Twenty (64.5%) out of 31 of ESBL-EC were ST131. PFGE revealed 29 different pulsotypes. CONCLUSIONS: Our study documented the high prevalence of ESBLs in E. coli isolates, with CTX-M-15 as the predominant ESBL gene. ST131 clone producing CTX-M-15 has a major presence in our hospital. The high prevalence of CTX-M producers was not due to the spread of a single clone. To the best of our knowledge, this study represents the first report of CTX-M-15 and CTX-M-27 ß-lactamases and the detection of the ST131 clone in Saudi E. coli isolates.

Distribution of ß-lactamases in carbapenem-non-susceptible Acinetobacter baumannii in Riyadh, Saudi Arabia.

In this study, the distribution of ß-lactamase genes among 55 consecutive Acinetobacter baumannii isolates with reduced susceptibility to imipenem collected at Prince Salman Hospital (Riyadh, Saudi Arabia) from February-June 2011 was investigated. Minimum inhibitory concentrations (MICs) were determined by Etest and were interpreted against Clinical and Laboratory Standards Institute (CLSI) breakpoints. PCR was used to search for ß-lactamase genes, insertion sequence ISAba1 and class 1 integrons. Imipenem MICs ranged from 2µg/mL to ≥32µg/mL and resistance to aztreonam, cefepime and ceftazidime was widespread, with MIC90 values (MIC required to inhibit 90% of the isolates) of >256µg/mL. blaTEM, blaADC and blaOXA-51-like genes were universal, whilst blaOXA-23, blaPER, blaGES and blaOXA-24 were found in 60.0%, 49.1%, 34.5% and 3.6% of isolates, respectively. Genes for SHV, CTX-M, VEB, KPC, OXA-58 and metallo-ß-lactamases (MBLs) were not detected. ISAba1 was universal and consistently present upstream of blaOXA-51, blaOXA-23, blaOXA-24 and blaADC; class 1 integrons also were universal. Notably, 28/55 isolates had both an extended-spectrum ß-lactamase (ESBLs) and an acquired blaOXA-23 gene. High-level carbapenem resistance (MIC≥32µg/mL) was consistently associated with blaOXA-23 or blaOXA-24, whereas low-level resistance (MIC of 2-8µg/mL) was associated with the presence of ESBLs of GES or PER type and/or ISAba1-upregulated blaOXA-51-like. In conclusion, blaTEM, blaOXA-23, blaPER and blaGES-like genes were prevalent, often in combination. MBLs remained absent and high-level carbapenem resistance consistently correlated with the presence of blaOXA-23 or blaOXA-24.

High prevalence of acquired quinolone-resistance genes among Enterobacteriaceae from Saudi Arabia with CTX-M-15 ß-lactamase.

We examined the prevalence of acquired quinolone resistance determinants among Enterobacteriaceae with extended-spectrum ß-lactamases (ESBLs) in Riyadh, Saudi Arabia. qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA genes were sought by polymerase chain reaction (PCR) in 160 nonduplicate, clinical Enterobacteriaceae with ESBLs from Prince Salman Hospital in Riyadh during 2009. MICs were determined for qnr- and aac(6')-Ib-cr-positive isolates. Mutations in gyrA and parC were determined for isolates with qnr. ESBL genes were characterized by PCR and sequencing. Among 99 ESBL-producing Escherichia coli, 73% were ciprofloxacin resistant, as were 74% of 61 ESBL-positive Klebsiella pneumoniae. The aac(6')-1b-cr gene was present in 76 ESBL producers, comprising 34 K. pneumoniae and 42 E. coli, whereas qnrA or qnrB genes were found in 21 isolates, all of them also harbouring aac(6')-1b-cr and bla(CTX-M-15), with the latter encoding what was considerably the dominant ESBL in the collection. The qnr-positive isolates harboured a variety of mutation in gyrA and parC but, even with aac(6')-1b-cr also present, high-level ciprofloxacin resistance (MIC >32 µg/mL) was invariably associated with double mutations in gyrA, affecting both Ser83 and Asp87 along with >1 substitution in parC, affecting Ser80 or Glu84.

Application of bleach method to improve sputum smear microscopy for the diagnosis of pulmonary tuberculosis.

In an active surveillance study, the bleach concentration method to improve sputum smear microscopy for the diagnosis of pulmonary tuberculosis was applied in chest symptomatics in Mahidpur block of Ujjain district in Madhya Pradesh, India. We purposely selected twenty villages with population of approximately 20,000 individuals. 664 sputum specimens from 297 chest symptomatics were collected. Ziehl-Neelsen staining was performed on direct sputum smears, smears made after bleach method and modified Petroff's method. Out of 297 chest symptomatics, 16 cases (5.38%) were positive by direct microscopy, 27 cases (9.09%) were positive by bleach method, 22 cases (7.40%) were positive by modified Petroff's method. The bleach method is safe, cheap, easy and sensitive. It can be applied for improved detection of Mycobacterium tuberculosis in hospitals and laboratories especially in settings where mycobacterial culture facilities are not available. The implementation of bleach method clearly improves case detection and can be a useful contribution in the National Tuberculosis Control Program.

Application of bleach method in diagnosis of extra-pulmonary tuberculosis.

Extra pulmonary tuberculosis (TB) comprises 15% of the total tuberculosis cases. Bleach concentration method for demonstration of Acid fast bacilli (AFB) has been recently described for sputum. The aim of this study is to apply this method for demonstration of AFB in material obtained from extra-pulmonary sites and to correlate with cytology and conventional Ziehl Neelsen (Z N) staining. A total of 55 samples were studied from clinically suspected cases of extrapulmonary TB which included, FNA lymph nodes (17), abscesses drained from various body parts (18), body fluids (18) and skin scrapping (2). All the samples were processed for routine cytology, conventional ZN staining and bleach method followed by ZN staining. Out of 55 samples, 24(43.40%) were indicative of tuberculosis on cytology, 12(21.8%) were positive for AFB on conventional ZN staining, while the positivity increased to 39(70.90%) by Bleach method. Bleach solution is inexpensive and readily available in hospitals and its application has been proved in pulmonary tuberculosis. However to the best of our knowledge this is a pioneer study applied to the extra-pulmonary samples and the results of the present study shows improved detection of AFB.