While rapid bacterial identification and susceptibility testing led to earlier changes and a significant reduction in antibiotic use, they did not reduce mortality.

A typing procedure for methicillin-resistant Staphylococcus aureus (MRSA) based on the polymerase chain reaction (PCR) amplification of both mecA sequences and variable DNA sequences as present in the prokaryotic genome has been developed. Two primers based on the sequences of DNA repeats as discovered in gram-negative members of the family Enterobacteriaceae allow detection of variable regions in the genome of a gram-positive bacterium such as S. aureus, as does a newly described arbitrary primer. This procedure, enabling the detection of 23 different genotypes in a collection of 48 MRSA isolates, was validated by comparisons with phage typing studies. It appeared that within the same group of isolates only 13 different phagovars could be identified. Combination of the results from both phage typing and genotyping allowed the discrimination of 34 of 48 isolates. However, depending on the primer-variable complexity of the PCR fingerprints, which could also be modulated by combination of PCR primers, clear homologies between the groups defined by either phage typing or fingerprinting were observed. An analysis of an MRSA outbreak in a geriatric institution showed a collection of genetically homogeneous isolates. In agreement with phage typing, PCR fingerprinting revealed the identical natures of the MRSA strains isolated from all patients.

An outbreak of methicillin-resistant Staphylococcus aureus (MRSA) involving 27 patients and 14 health-care workers (HCW) was studied. The outbreak started in the hematology unit of the University Hospital Rotterdam, Dijkzigt, The Netherlands, and spread to the surgical unit. Twenty-one patients (77.8%) developed clinical disease, and five died. Subsequently, MRSA was detected in food and in the throat of one of the HCW who prepared food for hematology patients. Food contaminated by an HCW most likely caused the first case of MRSA septicemia. This route of transmission has not been described before. The outbreak strain was probably transmitted to the surgical unit by a colonized nurse, where it caused an explosive outbreak. Airborne MRSA transmission played an important role in disseminating the organism. The outbreak was controlled within 6 months by intensifying surveillance, temporarily closing the affected wards, treating carriers, and instituting an MRSA ward outside the hospital. Phage typing, insertion sequence probing, protein A gene typing, and DNA fingerprinting by PCR revealed that all outbreak-related isolates were identical. By pulsedfield gel electrophoresis, all but one of the outbreak-related isolates were determined to be identical. Protein A gene typing identified numerous (11) repeat units in all outbreak-related isolates, which supports the suggestion that the outbreak strain may have been more virulent and more transmissible than other MRSA strains. Pheno-and genotyping studies underlined the value of DNA fingerprinting methods for investigation of MRSA epidemiology. Optimal discriminatory power was achieved by combining the results of four genotyping methods.

An international multicenter study was undertaken to investigate the epidemiological dynamics of penicillin-resistant pneumococci. We compared the molecular epidemiological characteristics of 205 penicillin-resistant isolates originating from The Netherlands, Thailand, United States, Spain, Greece, Poland, Cuba, Germany, Finland, United Kingdom, South Africa, Hungary, Portugal, Croatia, and the Czech Republic. Eighty-four distinct restriction fragment end labeling (RFEL) types were observed. Twenty-eight genetic types were shared by two or more strains. Five genetic clusters consisted of strains originating from different countries, illustrating dissemination of penicillin-resistant pneumococci among countries. The strains displaying the two predominant RFEL types corresponding with the pandemic clones 23F and 9V were found in 10 and 6 different countries, respectively. This clearly demonstrates the pandemic behavior of these two clones. Twelve out of the 28 genetic clusters contained two or more serotypes. This finding indicates frequent horizontal transfer of capsular genes. Within distinct RFEL types, identical penicillin binding protein (PBP) genotypes were often observed, suggesting a high frequency of horizontal transfer of penicillin resistance genes. The most predominant PBP type was found in 15 distinct RFEL types, comprised 44% of the entire collection, and was observed in 11 countries. The vast majority of the strains belonging to the pandemic clones 23F and 9V shared this predominant PBP type. We hypothesize that the clones 23F and 9V are responsible for the worldwide increase of penicillin-resistance, because they serve as a genetic reservoir for susceptible pneumococci to acquire penicillin resistance.

Typhoid fever is caused by Salmonella enterica serovar Typhi, a major public health concern in developing countries. Recently, there has been an upsurge in the occurrence of bacterial isolates that are resistant to ciprofloxacin, and the emergence of broad spectrum β-lactamases in typhoidal salmonellae constitutes a new challenge for the clinician. A total of 337 blood culture isolates of S. Typhi, isolated from Pondicherry, India, between January 2005 and December 2009, were investigated using phenotypic, molecular and serological methods. Of the 337 isolates, 74 (22%) were found to be multidrug resistant (MDR) and 264 (78%) nalidixic acid resistant (NAR). Isolates with reduced susceptibility to ciprofloxacin possessed single mutations in the gyrA gene. A high rate of resistance (8%) was found to ciprofloxacin. All isolates with a ciprofloxacin MIC ≥ 4 mg/L possessed both double mutations in the QRDR of the gyrA gene and a single mutation in the parC gene. Active efflux pump mechanisms were also found to be involved in ciprofloxacin resistance. Finally, a large number of PFGE patterns (non-clonal genotypes) were observed among the S. Typhi isolates. In conclusion, a high rate of ciprofloxacin resistance was observed in comparison to other endemic areas in blood culture isolates of S. Typhi from Pondicherry, India, with steadily increasing NAR but decreasing MDR isolations over the study period. This is most likely to be due to an increased use of ciprofloxacin as a first-line drug of choice over more traditional antimicrobial agents for the treatment of typhoid fever.