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Background The objective of this study was to determine the prevalence of plasmid-mediated AmpC (pAmpC) among Enterobacteriaceae isolated from humans and from retail meat in Egypt. Methods Enterobacteriaceae were isolated from patients with suspected bloodstream infection, human fecal samples, retail chicken meat samples and retail sheep meat samples. All group I Enterobacteriaceae were analyzed for presence of pAmpC genes by PCR. Antibiotic susceptibility testing was performed in all pAmpC positive isolates, followed by phenotypic and genotypic ESBL and carbapenemase testing on indication. Results The prevalence of pAmpC among group I Enterobacteriaceae isolated from 225 patients with bloodstream infection was 5.6% [95%CI 2.2–13.4]. Among 100 patients with community-onset gastroenteritis the prevalence in fecal samples was 4.8% [95%CI 2.1–10.7]. The prevalence among 112 chicken carcasses and 100 sheep meat samples was 2.4% [95%CI 0.7–8.4] and 1.1% [95%CI 0.2–5.7], respectively. In half of the AmpC positive isolates we detected an ESBL gene and 2 isolates harbored a carbapenemase gene. In five isolates there was resistance to at least three important alternative antibiotic drugs. Conclusions We consider the prevalence of pAmpC in Egypt, as found in our study, moderately low. To follow future trends in prevalence of pAmpC worldwide, a standardized screening algorithm for the detection of pAmpC is needed.

One unreported case of extended-spectrum-beta-lactamase (ESBL)-producing Salmonella enterica serovar Typhi was identified, whole-genome sequence typed, among other analyses, and compared to other available genomes of S . Typhi. The reported strain was similar to a previously published strain harboring bla SHV-12 from the Philippines and likely part of an undetected outbreak, the first of ESBL-producing S . Typhi.

In 271 Enterobacter blood culture isolates from 12 hospitals, extended-spectrum beta-lactamase (ESBL) prevalence varied between 0% and 30% per hospital. High prevalence was associated with dissemination, indicating the potential relevance of infection control measures. Screening with cefepime or Vitek 2, followed by a cefepime/cefepime-clavulanate Etest, was an accurate strategy for ESBL detection in Enterobacter isolates (positive predictive value, 100%; negative predictive value, 99%).

Sir, We thank Abecasis and colleagues for their comments on our paper on the detection of extended-spectrum b-lactamase (ESBL)-producing positive aerobic Gram-negative bacilli (ESBL-GNB) in patients on the intensive care unit (ICU) of our hospital, where selective decontamination of the digestive tract (SDD) is routinely used in mechanically ventilated patients. Their main comment is that the link that we suggest between SDD use and the detection of these strains is only circumstantial; they also argue that the occurrences of such isolates is related to their resistance to tobramycin, an antibiotic in the SDD regimen which could be replaced by neomycin or paromomycin. We agree that during treatment with SDD, ESBL-GNB isolates will only reach detectable numbers in cultures from patients if they have decreased susceptibility to tobramycin. We isolated four genetically different strains that carried identical plasmids, had identical resistance patterns, and carried identical ESBL genes. As we noted in our paper, the emergence of the four strains could be due to: (i) an increase in these strains from below to above detection level; (ii) acquisition of the strains carrying the resistance plasmid; or (iii) acquisition of the resistance plasmid by strains that belong to the normal flora of the patients. We do not state that the emergence of these strains would not have occurred if these patients had not received SDD; we only want to show that SDD does not prevent the emergence of such strains and possibly, like any other antimicrobial treatment including cephalosporins, even promotes their emergence. Possibility (i) is unlikely, as it implies that isolates with decreased cefotaxime and tobramycin susceptibility are frequently missed during screening in an SDD setting. Acquisition of strains with identical plasmids seems improbable because this presumes a widespread presence of plasmids with identical RFLP patterns in different isolates. RFLP analyses of plasmids isolated from two ESBL-positive and one ESBL-negative bacterial strains isolated during the same period in our ICU, showed different patterns. Since four independent events of acquisition of different strains with similar plasmids is unlikely, acquisition of the resistance plasmid by different strains seems the most likely explanation for our observation. The multidrug-resistant (MDR) strains described in our study were intermediately susceptible to polymyxin E. Of note, in our hospital, outside the ICU, polymyxin E is not usually used to treat infections caused by Escherichia coli or Klebsiella pneumoniae. Cefotaxime, the most common systemic antibiotic used in SDD regimens has been reported to be associated with the emergence of ESBL-producing strains and with an increase in ESBL prevalence. In our view, an important part of the problem is the colonization with strains carrying MDR plasmids, as every colonized patient forms a potential source which may facilitate further dissemination of ESBL genes. Although adjustment of the SDD regimen or isolation measures can eventually contain outbreaks, detection of ESBL plasmid-mediated outbreaks is more complex than that of clonal outbreaks. In all studies that evaluated emergence of resistance with SDD wherein cefotaxime is used, there was no optimal screening for ESBLs. Therefore, we suggest that for optimal evaluation of SDD regimens with respect to ESBL-producing strains and emergence of resistance, optimal screening techniques for detection of ESBLs is warranted. Obviously, optimal screening for ESBL production is also needed in ICUs where SDD is not used.

OBJECTIVES: During treatment with selective decontamination of the digestive tract (SDD), four multidrug-resistant (MDR) strains, three different Escherichia coli and one Klebsiella pneumoniae, were isolated from four patients not known as carriers of such MDR strains before their admission to the intensive care unit (ICU) in the Academic Medical Center (AMC) in Amsterdam. These isolates were extended-spectrum beta-lactamase (ESBL)-positive. We investigated whether this was due to interspecies transfer of resistance genes. METHODS: The MDR strains were typed by amplified fragment length polymorphism (AFLP) analysis. The plasmids from these strains were characterized by restriction fragment length polymorphism and the resistance genes were characterized by PCR and sequence analysis. RESULTS: The strains were genetically unrelated and contained identical plasmids with ESBL genes. CONCLUSIONS: We identified an outbreak of plasmid-mediated ESBL genes during SDD treatment in the ICU. The use of third-generation cephalosporins in SDD is associated with the emergence of ESBLs. We conclude that identification of emerging MDR Gram-negative bacteria and recognition of resistance plasmid transfer during SDD treatment are crucial for optimal application of this regimen in ICUs

SHV β-lactamases, encoded by blaSHV genes, confer resistance to a broad spectrum of β-lactam antimicrobial agents and are of significant therapeutic concern for infections caused by many species of gram-negative bacteria (4, 9, 11). At present, there are more than 50 varieties of SHV β-lactamases (7), the majority of which confer the extended-spectrum β-lactamase (ESBL) phenotype (10, 13, 14). Several molecular methods for the detection and differentiation of different SHV β-lactamases have been proposed, but nucleotide sequencing remains the standard for determination of the β-lactamase gene present in a strain (4). Although automated thermal cycling has improved sequence analysis of blaSHV genes (3), the quality of nucleotide sequencing data may vary, e.g., due to template quality. Usually, overall low-quality sequence data are easily identified and not used for further analysis, as are the low-quality starts and ends of sequence reads. However, quality values (e.g., Phred values [5]) are also assigned for individual positions by most sequence analysis software, but these are seldom reported. Minimal standards corresponding to a Phred value of 40 have been proposed for (human) whole-genome sequencing and for human clinical samples, but they are not uniformly applied to sequencing of bacterial targets. In this report we describe how careful interpretation and assessment of quality values prevented the misidentification of two different blaSHV alleles in single bacterial isolates. During a study on ESBL epidemiology (1), PCR products for β-lactamase genes of three ESBL-positive clinical strains of three different species, Escherichia coli, Pseudomonas aeruginosa, and Enterobacter cloacae, were amplified and sequenced with the Big Dye sequencing kit 1.1 (Applied Biosystems, Foster City, Calif.) as described previously (1). The sequence assembly of the individual sequence traces for each sample, generated with Codoncode Aligner 1.3.4 (CodonCode Corporation, Dedham, MA) using default parameters, yielded an SHV-2 gene for the E. coli and E. cloacae isolates and an SHV-5 gene for the P. aeruginosa isolate. Sequence assemblies of sequence traces of PCR products often show low-quality positions near the ends of the assembly. This was also the case in the present analyses. However, the present assemblies contained three additional low-quality positions (Fig. ​(Fig.1).1). Visual inspection of the individual trace files showed two overlapping signals at these three positions: A or T at position 92, A or G at position 402, and A or G at position 703 (Fig. ​(Fig.2A).2A). The polymorphisms at positions 92 and 703 lead to amino acid polymorphisms at positions 35 and 240 in the numbering scheme of Ambler and colleagues (2). The four possible different combinations correspond to SHV types SHV-2 (L35, E240), SHV-2a (Q35, E240), SHV-5 (L35, K240), and SHV-12 (Q35, K240) (http://www.lahey.org/Studies/), which differ in spectrum and activity. A possible explanation for the double signals could be that two or more blaSHV alleles were amplified from each isolate, resulting in a mixture of templates in the sequence reaction mixture. FIG. 1. Graphic representation of Phred quality values for the contig assembled from four separate trace files for one of the blaSHV PCR products. The quality value scale is logarithmic: a Phred value of 20 means a 1/100 likelihood of error, a Phred value of ... FIG. 2. Low-quality positions in sequence traces of a blaSHV PCR product. (A to C) Electropherograms of the regions containing nucleotide positions 92, 402, and 703. (A) Double signals in the PCR amplicon. (B) Signals in one class of clones, T92A402G703, corresponding ... To assess the number and type of blaSHV alleles in the PCR amplicons, the alleles were ligated into cloning vector pCR 2.1 (Invitrogen, Breda, The Netherlands). Ligation products were transformed to E. coli DH5α. Insert size was confirmed by colony PCR with the vector primers M13F (5′ GTA AAA CGA CGG CCA G 3′) and M13R (5′ CAG GAA ACA GCT ATG AC 3′). Each clone is the result of one individual DNA molecule from the population of blaSHV alleles in the PCR amplicon. Eight different clones for each of the PCR amplicons of the three different isolates were sequenced, and the signals at nucleotide positions 92, 402, and 703 were compared. Only two combinations of the three polymorphisms were detected in the 24 sequenced clones (Fig. 2B and C). A92G402A703, corresponding to SHV-2, was detected in four clones derived from the P. aeruginosa amplicon, five clones derived from the E. coli amplicon, and four clones from the E. cloacae amplicon. The remaining clones all contained the combination T92A402G703, corresponding to SHV-12. The distribution among the clones suggests that these two SHV genes were present in approximately identical copy numbers in each of the three isolates. The initial analysis of the sequence traces, based on the sequence assembly using default parameters, identified an SHV-2 gene in the E. coli and E. cloacae strains and an SHV-5 gene in the P. aeruginosa strain. This indicates that the use of default sequence assembly parameters without careful inspection of individual low-quality positions may lead to misidentification of the number and type of SHV genes carried by a single bacterial isolate. To our knowledge, this is the first report of two blaSHV genes in a single P. aeruginosa isolate. Only recently, the presence in a single E. cloacae isolate of two different blaSHV genes that differ by a single nucleotide, SHV-7 and SHV-30, was reported (12). Szabo and colleagues developed a method to detect this single-nucleotide polymorphism by fluorescence resonance energy transfer real-time PCR. Unfortunately, adapting this method to detect the SHV-2 and SHV-12 combination in the present isolates will be impossible, as these alleles contain three nucleotide polymorphisms, which theoretically give rise to four different combinations. The presence of the two β-lactamases in one strain has implications for its phenotype. The lysine residue at position 240 in SHV-12 is critical for the efficient hydrolysis of cefotaxime (6). In addition, the hydrolysis rate of aztreonam by SHV-12 is superior to that of SHV-2 (8). Therefore, proper identification of the SHV ESBL genes is important for optimal assessment of the cephalosporin sensitivity of a given strain. In addition, full understanding of the epidemiology of extended-spectrum β-lactamases requires accurate data on the presence of the different types of these enzymes in various species. Hence, careful evaluation of low-quality values (Fig. ​(Fig.11 and ​and2)2) in individual sequence traces is warranted. This is particularly relevant for positions where polymorphisms are known to cause changes in the spectrum of SHV β-lactamases, i.e., the positions corresponding to amino acid positions 35, 238, and 240 (4). Identification of multiple alleles can be crucial to identify outbreaks of resistance plasmids that carry ESBL genes and is essential to understand the dynamics and the evolution of these genes.

A transferable plasmid encoding SHV-12 extended-spectrum β-lactamase, TEM-116, and aminoglycoside resistance was responsible for two sequential clonal outbreaks of Enterobacter cloacae and Acinetobacter baumannii bacteria. A similar plasmid was present among isolates of four different bacterial species. Recognition of plasmid transfer is crucial for control of outbreaks of multidrug-resistant nosocomial pathogens.

Traditionally, bacteria in The Netherlands have low levels of resistance to antibiotics. This report describes what is believed to be the first carbapenem-resistant Klebsiella pneumoniae producing an OXA-48 type β-lactamase in The Netherlands. The isolate co-produced a CTX-M-15 type β-lactamase and was recovered from a patient who was transferred from a hospital in India to an intensive care unit in The Netherlands. His recovery in The Netherlands was complicated by pneumonia due to the carbapenem-resistant K. pneumoniae to which he eventually succumbed. Pre-emptive screening for carbapenem-resistant Enterobacteriaceae in selected patients could be imperative to maintain the low prevalence of these highly resistant bacteria in Dutch hospitals.

common mechanisms of fluoroquinolone resistance in Gramnegative organisms, whole-cell DNA of isolate 13 was extracted and used as a template for PCR amplification using primers able to amplify the corresponding quinolone resistance determining regions (‘QRDRs’) of the gyrA and parC genes of P. stutzeri ATCC 17588 (as control) and of P. stutzeri 13, and subsequently sequenced. The primers used were: GyrA-PstF (5 0 -CAT GGG CGA ACT GGC CAA AG-3 0 ) and GyrA-PstR (5 0 -CTG CCG GAA TCT GCT CGG TG-3 0 ) for gyrA (expected amplicon of 473 bp); and ParC-PstF (5 0 -ATG AGC GAA TCC CTC GAC CTG-3 0 ) and ParC-PstR (5 0 -GGT TCG TCC AGG GTG CCG TCG A-3 0 ) for parC (expected amplicon of

A Salmonella enterica serotype Typhi strain was cultured from blood and fecal samples from a 54-year-old man with fever and diarrhea. He had returned from travel to the Philippines a few days earlier. Phenotypic and genotypic analysis confirmed the production of the SHV-12 extended-spectrum beta-lactamase.