Organisms producing CTX-M-␤-lactamases are emerging around the world as a source of resistance to oxyiminocephalosporins such as cefotaxime (CTX). However, the laboratory detection of these strains is not well defined. In this study, a molecular detection assay for the identification of CTX-M-␤-lactamase genes was developed and used to investigate the prevalence of these enzymes among clinical isolates of Escherichia coli and Klebsiella species in the Calgary Health Region during 2000 to 2002. In addition, National Committee for Clinical Laboratory Standards (NCCLS) recommendations were evaluated for the ability to detect isolates with CTX-M extended-spectrum ␤-lactamases (ESBLs). The PCR assay consisted of four primer sets and demonstrated 100% specificity and sensitivity for detecting different groups of CTX-M-␤-lactamases in control strains producing well-characterized ESBLs. Using these primer sets, 175 clinical strains producing ESBLs were examined for the presence of CTX-M enzymes; 24 (14%) were positive for bla CTX-M-1-like genes, 95 (54%) were positive for bla CTX-M-14-like genes, and the remaining 56 (32%) were negative for bla CTX-M genes. Following the NCCLS recommendations for ESBL testing, all of the control and clinical strains were detected when screened with cefpodoxime and when both cefotaxime and ceftazidime with clavulanate were used as confirmation tests.Resistance to the expanded-spectrum cephalosporins can occur in Escherichia coli and Klebsiella species via the production of extended-spectrum ␤-lactamases (ESBLs) that are capable of hydrolyzing the oxyiminocephalosporins and monobactams (11). Recently, a family of ESBLs which preferentially hydrolyze cefotaxime (CTX), the CTX-M-␤-lactamases, have been recognized and reported in the literature with increasing frequency (3). This resistance mechanism is widespread throughout the world, with reports of clinical isolates producing these ␤-lactamases from Europe, Africa, Asia, South America, and most recently North America (3, 27).CTX-M-␤-lactamases are not closely related to TEM or SHV ESBLs (7) but share high amino acid identity with chromosomal ␤-lactamases from Kluyvera georgiana (34), Kluyvera cryocrescens (17) and Kluyvera ascorbata (24). In fact, the CTX-M-5 enzyme is identical to the chromosomal gene of K. ascorbata (3). According to a recent review and new data within GenBank, CTX-M-␤-lactamases can be divided into five groups based on their amino acid sequence identities (3). Group I includes CTX-M-1, -3, -10 to -12, -15 (UOE-1), -22, -23, -28, -29, and -30. Group II includes CTX-M-2, -4 to -7, and -20 and Toho-1. Group III includes CTX-M-8. Group IV includes CTX-M-9, -13, -14, -16 to -19, -21, and -27 and Toho-2. Finally group V includes CTX-M-25 and -26. The members of these groups exhibit Ͼ94% amino acid identity within the group and Յ90% amino acid identity between groups (3).The laboratory detection of organisms producing CTX-M-␤-lactamases is not well defined. The guidelines published by the National Committee for Clinical Labor...

Four isolates of Klebsiella pneumoniae obtained from patients at a Maryland medical centre exhibited reduced susceptibility to carbapenems and were found to produce the novel, class A, plasmid-mediated, carbapenem-hydrolysing enzyme, KPC-2. This enzyme has 99% identity with the plasmid-mediated, carbapenem-hydrolysing enzyme KPC-1, reported previously in a North Carolina K. pneumoniae isolate. The KPC-2-producing isolates were either susceptible or intermediate to imipenem and meropenem, unlike the KPC-1-producing isolate, which was resistant to these agents. Detection of KPC-2 may be a problem for clinical laboratories because in this study it was associated with positive extended-spectrum beta-lactamase (ESBL) confirmation tests (clavulanate-potentiated activities of ceftriaxone, ceftazidime, cefepime and aztreonam). Therefore, a failure to recognize the significance of reduced carbapenem susceptibility in the isolates that remained susceptible to imipenem or meropenem could have resulted in the isolates being incorrectly identified as ESBL producers.

Newer ␤-lactamases such as extended-spectrum ␤-lactamases (ESBLs), transferable AmpC ␤-lactamases, and carbapenemases are associated with laboratory testing problems of false susceptibility that can lead to inappropriate therapy for infected patients. Because there appears to be a lack of awareness of these enzymes, a study was conducted during 2001 to 2002 in which 6,421 consecutive, nonduplicate clinical isolates of aerobically growing gram-negative bacilli from patients at 42 intensive care unit (ICU) and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. From these isolates, 746 screenpositive isolates (11.6%) were referred to a research facility and investigated to determine the prevalence of ESBLs in all gram-negative isolates, transferable AmpC ␤-lactamases in Klebsiella pneumoniae, and carbapenemases in Enterobacteriaceae. The investigations involved phenotypic tests, isoelectric focusing, ␤-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular analyses. ESBLs were detected only in Enterobacteriaceae (4.9% of all Enterobacteriaceae) and were found in species other than those currently recommended for ESBL testing by the CLSI (formerly NCCLS). These isolates occurred at 74% of the ICU sites and 43% of the non-ICU sites. Transferable AmpC ␤-lactamases were detected in 3.3% of K. pneumoniae isolates and at 16 of the 63 sites (25%) with no difference between ICU and non-ICU sites. Three sites submitted isolates that produced class A carbapenemases. No class B or D carbapenemases were detected. In conclusion, organisms producing ESBLs and transferable AmpC ␤-lactamases were widespread. Clinical laboratories must be able to detect important ␤-lactamases to ensure optimal patient care and infection control.

Current food safety issues are deleteriously reshaping the life style of the population in the developing world. Socioeconomic status of the population in poorer economies is one of the major determinants to delineate the availability of safe food to the vulnerable population. Assessment of the prevalence of foodborne illness in developing world is the most neglected area to control disease. Botulism, Shigellosis, Campylobacteriosis, Escherichia coli infection, Staphylococcus aureus infection, Salmonellosis, Listeriosis and Cholerae are extensively prevalent and pose a major threat to human health in underdeveloped communities. The existing food safety status of many African, South Asian, Central, and South American developing countries is distressing therefore; it seems much timely to highlight the areas for the improvement to ensure the supply of safe food to the population in these regions. Extensive literature search at PubMed, Science Direct and Medline was carried out during the current year to catch on relevant data from 1976 to date, using selective terms like food safety, South East Asia, Africa, Central and South America, and foodborne illness etc. Efforts were made to restrict the search to low income countries of these regions with reference to specific foodborne pathogens. This report briefly discusses the present food safety situation in these developing countries and associated consequences as prime issues, suggesting foodborne illness to be the most distressing threat for human health and economic growth.

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory ganglionic neurons of infected animals. Expression of latency-related (LR) gene products is controlled by a 980-bp fragment (LR promoter).DNA sequence analysis revealed that two major open reading frames (ORFs) are in the LR gene. Antibodies directed against both ORFs were generated in rabbits by using synthetic peptides. Antibody P2, which is directed to sequences near the amino terminus of ORF 2, recognized a 41-kDa protein in lytically infected cells, suggesting that ORF 2 encodes a protein. When the LR gene was inserted into a mammalian expression vector and subsequently transfected into COS-7 cells, a 41-kDa protein was detected by use of silver-stained sodium dodecyl sulfate-polyacrylamide gels and by the P2 antibody. In contrast, this protein was not detected in mock-transfected cells. Deletion of DNA sequences containing ORF 2 blocked synthesis of the 41-kDa protein in COS-7 cells. Reverse transcriptase-mediated PCRs indicated that splicing occurs near the C terminus of ORF 2. Further studies indicated that LR RNA was alternatively spliced in latently infected cattle and that a fraction of LR RNA was poly(A) ؉ . Taken together, these studies suggested that a spliced LR transcript has the potential to encode a 41-kDa protein.

A strain of an Enterobacter sp. with reduced susceptibility to imipenem, which produced a plasmid-mediated class A carbapenem-hydrolyzing enzyme, KPC-2 β-lactamase, was isolated from a patient with sepsis at a Boston hospital. This is the first report of the production of a plasmid-encoded KPC-2 β-lactamase by an Enterobacter sp

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in the sensory ganglionic neurons of cattle. The exclusive viral RNA expressed in a latent infection is the latency-related (LR) RNA, suggesting that it regulates some aspect of a latent infection. During the course of a productive infection, alphaherpesviruses induce certain events which occur during cell cycle progression. Consequently, we hypothesized that a BHV-1 infection might induce events in neurons which occur during cell cycle progression. In agreement with this hypothesis, cyclin A was detected in neurons of trigeminal ganglia when rabbits were infected. Neuronal cell cycle progression or inappropriate expression of cyclin A leads to apoptosis, suggesting that a viral factor inhibits the deleterious effects of cyclin A expression. The BHV-1 LR gene inhibited cell cycle progression and proliferation of human osteosarcoma cells. Antibodies directed against cyclin A or the LR protein coprecipitated the LR protein or cyclin A, respectively, suggesting that the two proteins interact with each other. We conclude that LR gene products inhibit cell cycle progression and hypothesize that this activity enhances the survival of infected neurons.

The most commonly reported extended-spectrum ␤-lactamases (ESBLs) in U.S. isolates of Escherichia coli are TEM and SHV derived (2). CTX-M ESBLs have been reported elsewhere, mostly for clinical isolates of Salmonella enterica serovar Typhimurium, E. coli, and Klebsiella pneumoniae, but not in the United States (5). In this report we describe nine U.S. isolates of E. coli from five different states (Virginia, Idaho, Ohio, Washington, and Texas) that appear to produce CTX-M-like ESBLs. These isolates were discovered as we began to investigate the types of ESBLs produced by organisms in a U.S. hospital surveillance study. All isolates were obtained from patient specimens during 2001-2002. The origins of the isolates, pIs of the ␤-lactamases, and NCCLS broth microdilution MICs (4) are shown in Table 1. Pulsed-field gel electrophoresis (PFGE) results after restric